Collections of compounds

ABSTRACT

A compound of formula (IV): O is a solid support; L is a linking group or a single bond; X′ is selected from CO, NH, S, or O; A is O, S, NH, or a single bond; R 2  and R 3  are independently selected from: H, R, OH, OR, ═O, ═CH—R, ═CH 2 , CH 2 —CO 2 R′, CH 2 —CO 2 H, CH 2 —SO 2 R, O—SO 2 R, CO 2 R, COR, CN and there is optionally a double bond between C1 and C2 or C2 and C3; R 6 , R 7 , and R 9  are independently selected from H, R, OH, OR, halo, nitro, amino, Me 3 Sn; R 11  is either H or R; Q is S, O or NH; R 10  is a nitrogen protecting group; and Y is a divalent group such that HY=R, and other related compounds and collections of compounds.

This invention relates to collections of pyrrolobenzodiazepines, tomethods of synthesizing these compounds on solid supports, and tocompounds of utility therein. This invention further relates to methodsfor identifying and isolating pyrrolobenzodiazepine compounds withuseful and diverse activities from such collections.

BACKGROUND TO THE INVENTION

Compounds having biological activity can be identified by screeningcollections of compounds (i.e. libraries of compounds) produced throughsynthetic chemical techniques. Such screening methods include methodswherein the library comprises a plurality of compounds synthesized atspecific locations on the surface of a solid support where a receptor isappropriately labelled to identify binding to the compound, e.g.,fluorescent or radioactive labels. Correlation of the labelled receptorbound to the support with its location on the support identifies thebinding compound (U.S. Pat. No. 5,143,854).

Central to these methods is the screening of a multiplicity of compoundsin the library and the ability to identify the structures of thecompounds which have a requisite biological activity. In order tofacilitate synthesis and identification, the compounds in the libraryare typically formed on solid supports. Usually each such compound iscovalently attached to the support via a cleavable or non-cleavablelinking arm. The libraries of compounds can be screened either on thesolid support or as cleaved products to identify compounds having goodbiological activity.

BACKGROUND TO THE INVENTION

A large number of both synthetic and naturally occurring low molecularweight ligands are known that interact with DNA via a number ofdifferent mechanisms, including covalent or non-covalent interaction inthe minor or major grooves, intercalation between base pairs or othertypes of non-specific interactions.

A particular class of compounds which interacts with the minor grooveare the pyrrolobenzodiazepines (PBDs). PBDs have the ability torecognise and bond to specific sequences of DNA; the most preferredsequence is PuGPu (Purine-Guanine-Purine). The first PBD antitumourantibiotic, anthramycin, was discovered in 1965 (Leimgruber et al., 1965J. Am. Chem. Soc., 87, 5793-5795; Leimgruber et al., 1965 J. Am. Chem.Soc., 87, 5791-5793). Since then, a number of naturally occurring PBDshave been reported, and over 10 synthetic routes have been developed toa variety of analogues (Thurston et al., 1994 Chem. Rev. 1994, 433-465).Family members include abbeymycin (Hochlowski et al., 1987 J.Antibiotics, 40, 145-148), chicamycin (Konishi et al., 1984 J.Antibiotics, 37, 200-206), DC-81 (Japanese Patent 58-180 487; Thurstonet al., 1990, Chem. Brit., 26, 767-772; Bose et al., 1992 Tetrahedron,48, 751-758), mazethramycin (Kuminoto et al., 1980 J. Antibiotics, 33,665-667), neothramycins A and B (Takeuchi et al., 1976 J. Antibiotics,29, 93-96), porothramycin (Tsunakawa et al., 1988 J. Antibiotics, 41,1366-1373), prothracarcin (Shimizu et al., 1982 J. Antibiotics, 29,2492-2503; Langley and Thurston, 1987 J. Org. Chem., 52, 91-97),sibanomicin (DC-102)(Hara et al., 1988 J. Antibiotics, 41, 702-704; Itohet al., 1988 J. Antibiotics, 41, 1281-1284), sibiromycin (Leber et al.,1988 J. Am. Chem. Soc., 110, 2992-2993) and tomamycin (Arima et al.,1972 J. Antibiotics, 25, 437-444).

PBDs are of the general structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. There is either an imine (N═C), carbinolamine(NH—CH(OH))or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11position which is the electrophilic centre responsible for alkylatingDNA. All of the known natural products have an (S)-configuration at thechiral C11a position which provides them with a right-handed twist whenviewed from the C ring towards the A ring. This gives them theappropriate three-dimensional shape for isohelicity with the minorgroove of B-form DNA, leading to a snug fit at the binding site (Kohn,1975 In Antibiotics III. Springer-Verlag, New York, pp. 3-11; Hurley andNeedham-VanDevanter, 1986 Acc. Chem. Res., 19, 230-237). Their abilityto form an adduct in the minor groove enables them to interfere with DNAprocessing, hence their use as antitumour agents.

DISCLOSURE OF THE INVENTION

A first aspect of the present invention relates to compounds of formula(I):

wherein:

X is selected from COOH, NHZ, SH, or OH, where Z is either H or an amineprotecting group;

A is O, S, NH, or a single bond;

R₂ and R₃ are independently selected from: H, R, OH, OR, ═O, ═CH—R,═CH₂, CH₂—CO₂R, CH₁—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR and CN, and thereis optionally a double bond between C₁ and C₂ or C₂ and C₃;

R₆, R₇, and R₉ are independently selected from H, R, OH, OR, halo,nitro, amino, Me₃Sn;

R₁₁, is either H or R;

Q is S, 0 or NH;

R₁₀ is a nitrogen protecting group;

where R is a lower alkyl group having 1 to 10 carbon atoms, or anaralkyl group (i.e. an alkyl group with one or more aryl substituents),preferably of up to 12 carbon atoms, whereof the alkyl group optionallycontains one or more carbon-carbon double or triple bonds, which mayform part of a conjugated system, or an aryl group, preferably of up to12 carbon atoms; and is optionally substituted by one or more halo,hydroxy, amino, or nitro groups, and optionally contains one or morehetero atoms, which may form part of, or be, a functional group; and

Y is a divalent group such that HY=R.

These compounds are useful in the synthesis of collections ofpyrrolobenzodiazepines. Compounds of formula I can be attached to asolid support, e.g. via a connecting link which may comprise a chain ofcombinatorial units. Without N10-protection, the risk of undesirableside reactions with the imine bond during the coupling step would begreater.

If R is an aryl group, and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of, or be, a functional group,e.g. carbonyl, hydroxy.

R and HY groups are preferably independently selected from a lower alkylgroup having 1 to 10 carbon atoms, or an aralkyl group, preferably of upto 12 carbon atoms, or an aryl group, preferably of up to 12 carbonatoms, optionally substituted by one or more halo, hydroxy, amino, ornitro groups. It is more preferred that R and HY groups areindependently selected from lower alkyl groups having 1 to 10 carbonatoms optionally substituted by one or more halo, hydroxy, amino, ornitro groups. It is particularly preferred that R or HY areunsubstituted straight or branched chain alkyl groups, having 1 to 10,preferably 1 to 6, and more preferably 1 to 4, carbon atoms, e.g.methyl, ethyl, propyl, butyl.

Alternatively, R₆, R₇, and R₉, may preferably be independently selectedfrom R groups with the following structural characteristics:

(i) an optionally substituted phenyl group;

(ii) an optionally substituted ethenyl group;

(iii) an ethenyl group conjugated to an electron sink. The term‘electron sink’ means a moiety covalently attached to a compound whichis capable of reducing electron density in other parts of the compound.Examples of electron sinks include cyano, carbonyl and ester groups.

The term ‘nitrogen protecting group’ (or ‘amine protecting group’) hasthe meaning usual in synthetic chemistry, particularly synthetic peptidechemistry. It means any group which may be covalently bound to thenitrogen atom of the pyrrolobenzodiazepine (or amine) grouping, andpermits reactions to be carried out upon the molecule containing thisgrouping without its removal. Nevertheless, it is able to be removedfrom the nitrogen atom without affecting the remainder of the molecule.Suitable nitrogen protecting groups for the present invention includeFmoc (9-fluorenylmethoxycarbonyl), Nvoc (6-nitroveratryloxycarbonyl),Teoc (2-trimethylsilylethyloxycarbonyl), Troc(2,2,2-trichloroethyloxycarbonyl), Boc (t-butyloxycarbonyl), CBZ(benzyloxycarbonyl), Alloc (allyloxycarbonyl), and Psec(2(-phenylsulphonyl)ethyloxycarbonyl). Other suitable groups aredescribed in Protective Groups in Organic Synthesis, T Green and P Wuts,published by Wiley, 1991, which is incorporated herein by reference. Itis preferred that the nitrogen protecting group has a carbamatefunctionality where it binds to the nitrogen atom at the 10 position ofthe PBD ring structure.

R₇ is preferably an electron donating group. ‘Electron donating group’means a moiety covalently attached to a compound which is capable ofincreasing electron density in other parts of the molecule. Examples ofelectron donating groups useful in the present invention include alkyl,amine, hydroxyl, alkoxy and the like.

In compounds of formula I, Q is preferably O, and R₁₁ is preferably H.Independently, R₆ and R₉ are preferably H, and R₇ is preferably analkoxy group, and more preferably methoxy or ethoxy. It is furtherpreferred that if there is a double bond in the C ring, it is between C2and C3. In this case, R₂ and R₃ are preferably H.

—Y—A— is preferably an alkoxy chain, preferably an ethoxy chain.

A second aspect of the present invention relates to compounds of formulaII:

wherein

Y, A, R₇, R₂, R₃, R₆, and R₉ are as defined in the first aspect of theinvention;

X′ is CO, NH, S or O;

T is a combinatorial unit;

and n is a positive integer, where if n is greater than 1, each T may bedifferent.

It is preferred that X′ is either CO or NH. n may preferably be from 1to 16, and more preferably from 3 to 14.

A third aspect of the present invention relates to compounds of theformula III:

wherein

X′, Y, A, R₇, R₂, R₃ , R₆, R₉, and T are as defined in the second aspectof the invention;

n is zero or a positive integer;

L is a linking group, or less preferably a single bond;

and ◯ is a solid support, where if n is greater than 1, each T may bedifferent.

A fourth aspect of the present invention relates to compounds of theformula IV:

wherein X′, Y, A, R₇, R₂, R₃, R₆, R₉,T, n, L and ◯ are as defined in thethird aspect of the invention, and R₁₀, R₁₁, and Q are as defined in thefirst aspect of the invention.

A fifth aspect of the present invention relates to a method of makingcompounds of formula IV as described in the fourth aspect of theinvention by reacting compounds of formula I with compounds of formulaV:

wherein ◯, L, T and n are as defined in the fourth aspect of theinvention, and B is H or an atom or group for providing a functionalgroup capable of reaction with X.

A sixth aspect of the present invention relates to compounds of formulaVI:

wherein

◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉ and T are as defined in the secondaspect of the invention;

n and m are positive integers, or one of them may be zero;

T′ is a combinatorial unit, where each T′ may be different if m isgreater than 1;

T″ is a combinatorial unit which provides a site for the attachment ofX′; and

p is a positive integer, where if p is greater than 1, for eachrepeating unit, the meaning of X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″and the values of n and m are independently selected.

For example, if X′ is CO then the site on T″ may be NH, and if X′ is NH,S or O, then the site on T″ may be CO.

In a preferred aspect of the sixth aspect of the present invention, thecompound is of formula (VIa):

wherein ◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T″ are as defined above.

A seventh aspect of the present invention relates to compounds offormula VII:

wherein ◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″, n, m and p are asdefined in the sixth aspect and Q, R₁₀, and R₁₁, are as defined in thefirst aspect of the invention, where if p is greater than 1, for eachrepeating unit the meanings of X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″,Q, R₁₀ , R₁₁ and the values of n and m are independently selected.

An eighth aspect of the present invention relates to compounds offormula VIII:

wherein X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″, n, m and p are asdefined in the sixth aspect of the invention, where if p is greater than1, for each repeating unit the meanings of X′, Y, A, R₂, R₃, R₆, R₇, R₉,T, T′, T″ and values of n and m are independently selected.

A ninth aspect of the present invention relates to compounds of formulaIX:

wherein X′, Y, A, R₂, R₃, R₆, R₇, R₉, Q, R₁₀, R₁₁, T, T′, T″, n, m and pare as defined in the seventh aspect of the invention, where if p isgreater than 1, for each repeating unit the meanings of X′, Y, A, R₇,R₂, R₃, R₆, R₉, T, T′, T″,Q, R₁₀, R₁₁ and values of n and m areindependently selected.

A tenth aspect of the present invention relates to compounds of formulaX:

wherein ◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″, n, m and p are asdefined in the sixth aspect of the invention, and X″, Y′, A′, R′₂, R′₃,R′₆, R′₇ and R′₉ are selected from the same possibilities as X′, Y, A,R₂, R₃, R₆, R₇ and R₉ respectively, and where if p is greater than 1,for each repeating unit the meaning of X′, Y, A, R₂, R₃, R₆, R₇, R₉, T,T′, T″ and the values of n and m may be independently selected.

An eleventh aspect of the present invention relates to compounds offormula XI:

wherein ◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉, X″, Y′, A′, R′₂, R′₃, R′₆,R′₇, R′₉, T, T′, T″, n, m and p are as defined in the tenth aspect ofthe invention, Q, R₁₀, and R₁₁, are as defined in the first aspect ofthe invention, and Q′, R′₁₀, R′₁₁, have the same definitions as Q, R₁₀,R₁₁, respectively, and where if p is greater than 1, for each repeatingunit the meanings of X′, Y, A, R₂, R₃, R₆ , R₇, R₉, T, T′, T″, Q, R₁₀,R₁₁ and the values of n and m are independently selected.

A twelfth aspect of the present invention relates to compounds offormula XII:

wherein X′, Y, A, R₇, R₂, R₃, R₆, R₉, X″, Y′, A′, R′₇, R′₂, R′₃, R′₆,R′₉, T, T′, T″, n, m and p are as defined in the tenth aspect of theinvention, and where if p is greater than 1, for each repeating unit themeanings of X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, and T″ and the valuesof n and m may be independently selected.

A thirteenth aspect of the present invention relates to compounds offormula XIII:

wherein X′, Y, A, R₂, R₃, R₆, R₇, R₉, Q, R₁₀, R₁₁, X″, Y′, A′, R′₂, R′₃,R′₆, R′₇, R′₉, Q′, R′₁₀, R′₁₁, T, T′, T″, n, m and p are as defined inthe eleventh aspect of the invention, and where if p is greater than 1,for each repeating unit the meanings of X′, Y, A, R₂, R₃, R₆, R₇, R₉, T,T′, T″, Q, R₁₀, R₁₁ and the values of n and m may be independentlyselected.

A fourteenth aspect of the present invention relates to compounds offormula XIV:

wherein ◯, L, X′, Y, A, R₂, R₃, R₆, R₇, R₉, T, T′, T″, n, m and p are asdefined in the sixth aspect of the invention, and T′″ and q are selectedfrom the same possibilities as T and n respectively, and where if p isgreater than 1, for each repeating unit the meaning of T, T′, T″, T′″and the values of n, m and q may be independently selected.

A fifteenth aspect of the present invention relates to compounds offormula XV:

wherein ◯, L, X′, Y, A, R₂ , R₃, R₆, R₇, R₉, T, T′, T″, T″′, n, m, p andq are as defined in the fourteenth aspect of the invention, Q, R₁₀, andR₁₁, are as defined in the first aspect of the invention, and where if pis greater than 1, for each repeating unit the meanings of T, T′, T″,T″′and the values of n, m and q may be independently selected.

A sixteenth aspect of the present invention relates to compounds offormula XVI:

wherein X′, Y, A, R₇, R₂, R₃, R₆, R₉,T, T′, T″, T′″, n, m, p and q areas defined in the fourteenth aspect of the invention, and where if p isgreater than 1, the meanings of T, T′, T″, T″′ and values of n, m and qmay be independently selected.

A seventeenth aspect of the present invention relates to compounds offormula XVII:

wherein X′, Y, A, R₂, R₃, R₆, R₇, R₉, Q, R₁₀, R₁₁, T, T′, T″, T″′, n, m,p and q are as defined in the fourteenth aspect of the invention, andwhere if p is greater than 1, for each repeating unit the meanings of T,T′, T″, T″′ and the values of n, m and q may be independently selected.

Solid Support

The term ‘solid support’ refers to a material having a rigid orsemi-rigid surface which contains or can be derivatized to containreactive functionalities which can serve for covalently linking acompound to the surface thereof. Such materials are well known in theart and include, by way of example, silicon dioxide supports containingreactive Si-OH groups, polyacrylamide supports, polystyrene supports,polyethyleneglycol supports, and the like. Such supports will preferablytake the form of small beads, pins/crowns, laminar surfaces, pellets,disks. Other conventional forms may also be used.

Linker Group

One class of linking groups suitable for the present application is onewhich provide in the structure:

at least one covalent bond which can be readily broken by specificchemical reactions (or by light or changes in pH) thereby providing forliberation of compounds free from the solid support. The methodsemployed to break the covalent bond are selected so as to be specificfor the desired bond breakage thereby preventing unintended reactionsfrom occurring elsewhere on the complex. The linking group is selectedrelative to the synthesis of the compounds to be formed on the solidsupport so as to prevent premature cleavage of this compound from thesolid support as well as to limit interference by any of the proceduresemployed during compound synthesis on the support.

Examples of resins incorporating linking groups are set out in the tablebelow, which also indicates the groups that can be immobilised thereon,along with the suggested cleavage methods for the linking group. Suchresins are commercially available (e.g. from NovaBiochem). The tablealso indicates which of the linker groups are suitable for compoundswhere the N10 position is not protected, i.e. where the cleavage methodswould not affect an N10-C11 imine bond.

Compatible with Linker/Resin Cleavage Non-Protected Type ImmobilisesMethod PBD 2-Chlorotrityl RNH₂, RCO₂H,  1‥50% TFA Possibly chloride ROH,RSH Trityl chloride RNH₂, RCO₂H,  1-5% TFA Yes ROH, RSH 2-MethoxytritylRNH₂, RCO₂H,  1-5% Yes chloride ROH, RSH Rink amide RCO₂H 95% TFA Yesresin Sieber amide RCO₂H  1% TFA Yes resin 4-Sulfamyl- RCO₂H Alkylation/YES benzoyl amines Wang resin ROH, ArOH, 15-95% TFA Possibly RNH₂, RCO₂Hor DDQ or CAN HMPB-BHA ROH, ArOH,  1% TFA Possibly RCO₂H BromoethylRNH₂, RCO₂H, hν YES photolinker ROH, RSH Hydroxy ethyl RCO₂H hν YESphotolinker Aminoethyl RCO₂H hν YES photolinker

For protected PBDs the most preferred linking group is the Rink linker,which is cleavable by TFA. The N-protected PBDs can then be deprotectedusing photolysis. For unprotected PBDs the linking groups of choice arethose which are photolabile.

It is also possible that the linking group is a simple functionalityprovided on the solid support, e.g. amine, and in this case the linkinggroup may not be readily cleavable. This type of linking group is usefulin the synthesis of large split and mix libraries which will besubjected to on-bead screening (see below), where cleavage isunnecessary. Such resins are commercially available from a large numberof companies including NovaBiochem, Advanced ChemTech and Rapp Polymere.These resins include amino-Tentagel, and amino methylated polystyreneresin.

Combinatorial Unit

The term ‘combinatorial unit’ means any monomer unit which can be usedto build a chain attached to the solid support, usually by a linkinggroup. Examples of molecules suitable for such chain building are foundin Schreiber et al. (JACS, 120. 1998, pp.23-29), which is incorporatedherein by reference. An important example of a unit is an amino acidresidue. Chains may be synthesised by means of amine-protected aminoacids. Fmoc protected amino-acids are available from a number ofsources, such as Sigma and Nova Biochem. Both natural and unnaturalamino acids can be used, e.g. D- and L-amino acids and heterocyclicamino acids. In particular, heterocyclic amino acids of the type foundin the construction of netropsin and distamycin are of interest becauseof their DNA-recognition properties.

Amine units can be used to make up peptoids: see Soth, M. J. and Nowick,J. S. 1997, Unnatural oligomer libraries, Curr. Opin, Chem. Biol. 1, no.1: 120-129; Zuckermann et al., 1994, Discovery of Nanomolecular Ligandsfor 7-Transmembrane G-Protein-Coupled Receptors from a DiverseN-(Substituted)glycine Peptoid Library, Journal of Medicinal Chemistry37: 2678-85; Figliozzi, GMR et al., 1996, Synthesis of N-substitutedGlycine Peptoid Libraries, Methods in Enzymology, 267: 437-47; Simon, RJ et al. , 1992, Peptoids: A Modular Approach to Drug Discovery, Proc.Natl. Acad. Sci. USA, 89:9367-71; which are all incorporated herein byreference.

Other combinatorial units include PNAs (peptidonucleic acids): P ENielsen, et al, Science, 1991, 254, 1497; M Egholm, et al, Nature, 1993,365, 566; M Egholm et al, JACS, 1992, 114, 1895; S C Brown, et al,Science, 1994, 265, 777; 5. K Saha, et al, JOC, 1993, 58, 7827;oligoureas: Burgess K, et al, 1995, Solid Phase Synthesis of UnnaturalBiopolymers Containing Repeating Urea Units. Agnew. Chem. Int. ExaminingDivision. Engl 34, no. 8:907; Burgess K, et al, 1997, Solid PhaseSynthesis of Oligoureas; Journal of the American Chemical Society 119:1556-64; and oligocarbamates: Moran E J et al, 1995, Novel Biopolymersfor Drug Discovery. Biopolymers (Peptide Science); John Wiley and Sons37: 213-19; Cho C Y et al, 1993, An unnatural Biopolymer. Science 261:1303-5; Paikoff S F et al, 1996, The Solid Phase Synthesis ofN-Alkylcarbamate Oligomers. Tetrahedron Letters 37, no. 32: 5653-56. Allof these documents are incorporated herein by reference.

A type of combinatorial unit of particular relevance to the presentinvention is one based on the pyrrolobenzodiazepine structures; theseare of general formulae XVIIIa and XVIIIb:

wherein R₃, R₆, R₇, R₉, R₁₀, R₁₁, Q, A and Y are as defined in the firstaspect of the invention, and A′, and Y′ are independently selected fromthe possible groups for A and Y respectively.

A further type of particularly relevant combinatorial unit is one basedon a cyclopropyl indole (“a CPI unit”). Such units are known to interactcovalently with the minor groove of DNA, being specific for AT. Theseunits are of general formulae XIXa and XIXb:

wherein

P_(x) (if present) is an electrophilic leaving group;

P_(y) (if present) is selected from NH-Prot, O-Prot, S-Prot, NO₂, NHOH,N₃, NHR, NRR, N═NR, N(O)RR, NHSO₂R, N═NPhR, SR or SSR, where Protrepresents a protecting group;

P′_(y) (if present) is selected from NH, O and S;

D and E collectively represent a fused benzene or pyrrole ring (ineither orientation), which is optionally substituted by up torespectively 3 or 1 additional groups independently selected from R, OH,OR, halo, nitro, amino, Me₃Sn, CO₂H, CO₂R; P₂ and P₇ are independentlyselected from H, R, OH, OR, halo, nitro, amino, Me₃Sn.

The preferences for R are as above. P_(y) is preferably NH-Prot, O-Prot,S-Prot and P_(x) is preferably halogen or OSO₂R. It is further preferredthat the —CO₂— substituent is in the 2 or 3 position of the benzene ringor the 2 position of the pyrrole ring. P₂ and P₇ are preferably H.

These compounds may be synthesised using the techniques described byBoger et al, Chem. Rev. 1997, 97, 787-828; Cava et al, Drost, K. J.;Cava, M. P. J. Org. Chem. 1991, 56, 2240-2244; Rawal, V. H.; Jones, R.J.; Cava, M. P. J. Org. Chem. 1987, 52, 19-28; and Aristoff J. Med.Chem. 1993, 1992, 57, 6234-6239.

The following synthesis is provided as an example of the application ofthe Boger method.

Synthesis of a CPI Combinational Unit

The synthesis starts with a Wadsworth-Horner-Emmons condensation of3-bromo-benzaldehyde with the Sargent phosphonate which predominantlyprovides the E-isomer, which in turn undergoes acid-catalyzeddeprotection and Friedel-Crafts acylation. This generates thefunctionalised precursor, which is followed by 5-exo-trig arylradical-alkene cyclization.

Reagents and conditions: a: NaH, Sargent phosphate; b; TFA; c: 1)Ac₂O—KOAc; 2) K₂CO₃; 3) BnBr, K₂CO₃; d: CuCN; e: 1) LiOh; 2) DPPA,t-BuOH; f: NIS; g: allyl Br, NaH; h: Bu₃SnH, TEMPO; i: Zn—HOAc; j:Ph₃P—CCl; k: NaOH.

Aromatic nucleophilic substitution, ester hydrolysis and Curtiusrearrangements effected by treatment with DPPA are followed byregioselective C4 iodination and N-alkylation with allyl bromide. Thearyl radical-alkene cyclization by means of TEMPO as radical trap, asdescribed in Boger synthesis of CBI, provides the tricyclic system that,after conversion to the primary chloride and base-catalized hydrolysisof the cyano group, gives the desired combinatorial unit. The presentinvention relates to libraries, or collections, of compounds all ofwhich are represented by a single one of the formulae I to IV, and VI toXVII. The diversity of the compounds in a library may reflect thepresence of compounds differing in the identities of one or more of thesubstituent groups and/or in the identities of the combinatorial units T(when present). The number of members in the library depends on thenumber of variants, and the number of possibilities for each variant.For example, if it is the combinatorial units which are varied, andthere are 3 combinatorial units, with 3 possibilities for each unit thelibrary will have 27 compounds. 4 combinatorial units and 5possibilities for each unit gives a library of 625 compounds. If forinstance there is a chain of 5 combinatorial units with 17 possibilitiesfor each unit, the total number of members in the library would be 1.4million. A library may therefore comprise more than 1 000, 5 000, 10000, 100 000 or a million compounds, which may be arranged as describedbelow.

In the case of free compounds (formulae I, II, VIII, IX, XII, XIII, XVI,and XVII) the individual compounds are preferably in discrete volumes ofsolvents, e.g. in tubes or wells. In the case of bound compounds(formulae III, IV, VI, VII, X, XI, XIV and XV) the individual compoundsare preferably bound at discrete locations, e.g. on respectivepins/crowns or beads. The library of compounds may be provided on aplate which is of a suitable size for the library, or may be on a numberof plates of a standard size, e.g. 96 well plates. If the number ofmembers of the library is large, it is preferable that each well on aplate contains a number of related compounds from the library, e.g. from10 to 100. One possibility for this type of grouping of compounds iswhere only a subset of the combinatorial units, or substituents, areknown and the remainder are randomised; this arrangement is useful initerative screening processes (see below). The library may be presentedin other forms that are well-known.

A further aspect of the present invention is a method of preparing adiverse collection, or library of compounds as discussed above. If thediversity of the library is in the combinatorial units, then the librarymay be synthesised by the stepwise addition of protected combinatorialunits to a PBD core, each step being interposed by a deprotection step.Such a method is exemplified later. Libraries of this type can beprepared by the method known as “split and mix” which is described inFurka, A; Sebestyen, F; Asgedom, M and Dibo, G; General Method of RapidSynthesis of Multicomponent Peptide Mixtures; International Journal ofPeptide and Protein Research; 1991, 37, 487-193, which is incorporatedherein by reference. If the diversity of the library is in thesubstituent groups, the library may be synthesised by carrying out thesame synthetic methods on a variety of starting materials or keyintermediates, which already possess the necessary substituent patterns.

The present invention also relates to a method of screening thecompounds of formula II, III, VI, VIII, X, XII, XIV and XVI to discoverbiologically active compounds. The screening can be to assess thebinding interaction with nucleic acids, e.g. DNA or RNA, or proteins, orto assess the affect of the compounds against protein-protein or nucleicacid-protein interactions, e.g. transcription factor DP-1 with E2F-1, orestrogen response element (ERE) with human estrogen receptor (a 66 kdprotein which functions as hormone-activated transcription factor, thesequence of which is published in the art and is generally available).The screening can be carried out by bringing the target macromoleculesinto contact with individual compounds or the arrays or librariesdescribed above, and selecting those compounds, or wells with mixturesof compounds, which show the strongest effect.

This effect may simply be the cytotoxicity of the compounds in questionagainst cells or the binding of the compounds to nucleic acids. In thecase of protein-protein or nucleic acid-protein interaction, the effectmay be the disruption of the interaction studied.

The binding of the compounds to nucleic acids may be assessed bylabelling oligomers which contain a target sequence, and measuring theamount of labelled oligomers that bind to the compounds tested. Thelabelling may either be radio-labelling, or alternatively be labelsdetectable under visible or ultra-violet light. If this latter form ofscreening is carried out on compounds bound to solid supports which arein separate locations, the screening for results can be carried outvisually under a microscope. A similar technique is described in detailin “DNA-Binding ligands from peptide libraries containing unnaturalamino acids”, Lescrinier et al., Chem Eur J, 1998, 425-433. Thesetechniques are particularly suited to a one-step screening of a completelibrary of compounds, especially a large library made by the “split andmix” method described above.

Protein-protein interactions can be measured in a number of ways, e.g.FRET (fluorescence resonance energy transfer) which involves labellingone of the proteins with a fluorescent donor moiety and the other withan acceptor which is capable of absorbing the emission from the donor;the fluorescence signal of the donor will be altered depending on theinteraction between the two proteins. Another method of measuringprotein-protein interactions is by enzymatic labelling, using, forexample, horseradish peroxidase.

The screening process may undergo several iterations by selecting themost active compounds, or group of compounds, tested in each iteration;this is particular useful when testing arrays of wells which includemixtures of related compounds. Furthermore, if the wells containcompounds for which only a subset of the combinatorial units, orsubstituents, are known, but the rest are randomised, subsequentiterations can be carried out by synthesising compounds possessing theselected known (and successful) combinatorial unit, or substituent,pattern, but with further specified combinatorial units, orsubstituents, replacing the previously randomised combinatorial units,or substituents, adjacent the already known pattern; the remainingcombinatorial units, or substituents, are randomised as in the previousiteration. This iterative method enables the identification of activemembers of large libraries without the need to isolate every member ofthe library.

A further feature of this aspect is formulation of selected compound orcompounds with pharmaceutically acceptable carriers or diluents.

In yet further aspects, the invention provides a pharmaceuticalcomposition comprising a compound of formula II, VIII, XII or XVI and apharmaceutically acceptable carrier or diluent; and the use of acompound of formula II, VIII, XII or XVI in the manufacture of amedicament for the treatment of a gene-based disease, or a bacterial,parasitic or viral infection. Gene-based disease include neoplasticdisease, and Alzheimer's disease, and also include any diseasesusceptible to regulation of gene-expression.

Compounds of formula II, VIII, XII or XVI may be used in a method oftherapy against a gene-based disease, such as cancer or Alzheimer'sdisease, or a viral, parasitic or bacterial infection.

Another aspect of the present invention relates to the use of compoundsof formula III, VI, X or XIV in diagnostic methods. A compound offormula III, VI, X, XIV which binds to an identified sequence of DNA ora protein known to be an indicator of a medical condition can be used ina method of diagnosis. The method may involve passing a sample, e.g. ofappropriately treated blood or tissue extract, over an immobilisedcompound of formula III, VI X, XIV, for example in a column, andsubsequently determining whether any binding of target DNA to thecompound of formula III, VI or X has taken place. Such a determinationcould be carried out by passing a known amount of labelled target DNAknown to bind to compound III, VI or X through the column, andcalculating the amount of compound III, VI, X or XIV that has remainedunbound.

A further aspect of the present invention relates to the use ofcompounds of formula II, VIII, XII or XVI in target validation. Targetvalidation is the disruption of an identified DNA sequence to ascertainthe function of the sequence, and a compound of formula II, VIII, XII orXVI can be used to selectively bind an identified sequence, and thusdisrupt its function, i.e. functional genomics

Preferred Synthetic Strategies

A key step in a preferred route to compounds of formula I is acyclisation process to produce the B-ring, involving generation of analdehyde (or functional equivalent thereof) at what will be the11-position, and attack thereon by the pro-10-nitrogen:

In this structure, D represents XY, or a masked form thereof. The“masked aldehyde” —CPQ may be an acetal or thioacetal, in which case thecyclisation involves unmasking. Alternatively, the masked aldehyde maybe an aldehyde precursor, such as an alcohol —CHOH, in which case thereaction involves oxidation, e.g. by means of TPAP or DMSO (Swernoxidation).

The masked aldehyde compound can be produced by condensing acorresponding 2-substituted pyrrolidine with a 2-nitrobenzoic acid:

The nitro group can then be reduced to —NH₂ and protected by reactionwith a suitable agent, e.g. a chloroformate, which provides theremovable nitrogen protecting group in the compound of formula I.

A process involving the oxidation-cyclization procedure is illustratedin scheme 1 (an alternative type of cyclisation will be described laterwith reference to scheme 2).

If R₁₁ is other than hydrogen, the compound of formula I, may beprepared by direct etherification of the alcohol Ia. Compounds with Q=Scan be prepared by treatment of the corresponding alcohol Ia with R₁₁SH,and a catalyst (usually an acidic solution HCI, and sometimes a LewisAcid such as Al₂O₃). If Q=NH, then these compounds can be prepared byreacting Ia with an amine R₁₁NH and a catalyst (usually an aqueous acidor a Lewis Acid).

Exposure of the alcohol B (in which the 10-nitrogen is generallyprotected as a carbamate) to tetrapropylammonium perruthenate(TPAP)/N-methylmorpholine N-oxide (NMO) over A4 sieves results inoxidation accompanied by spontaneous B-ring closure to afford thedesired product. The TPAP/NMO oxidation procedure is found to beparticularly convenient for small scale reactions while the use ofDMSO-based oxidation methods, particularly Swern oxidation, provessuperior for larger scale work (e.g. >1 g).

The uncyclized alcohol B may be prepared by the addition of a nitrogenprotecting reagent of formula D, which is preferably a chloroformate oracid chloride, to a solution of the amino alcohol C, generally insolution, generally in the presence of a base such as pyridine(preferably 2 equivalents) at a moderate temperature (e.g. at 0° C.).Under these conditions little or no O-acylation is usually observed.

The key amino alcohol C may be prepared by reduction of thecorresponding nitro compound E, by choosing a method which will leavethe rest of the molecule intact. Treatment of E with tin (II) chloridein a suitable solvent, e.g. refluxing methanol, generally affords, afterthe removal of the tin salts, the desired product in high yield.

Exposure of E to hydrazine/Raney nickel avoids the production of tinsalts and may result in a higher yield of C, although this method isless compatible with the range of possible C and A-ring substituents.For instance, if there is C-ring unsaturation (either in the ringitself, or in R₂ or R₃), this technique may be unsuitable.

The nitro compound of formula E may be prepared by coupling theappropriate o-nitrobenzoyl chloride to a compound of formula F, e.g. inthe presence of K₂CO₃ at −25° C. under a N₂ atmosphere. Compounds offormula F can be readily prepared, for example by olefination of theketone derived from L-trans-4-hydroxy proline. The ketone intermediatecan also be exploited by conversion to the enol triflate for use inpalladium mediated coupling reactions.

The o-nitrobenzoyl chloride is synthesised from the o-nitrobenzoic acid(or, after hnydrolysis, the alkyl ester) of formula G, which itself isprepared from the vanillic acid (or alkyl ester) derivative H. Many ofthese are commercially available and some are disclosed in Althuis, T.H. and Hess, H. J., J. Medicinal Chem, 20(1), 146-266 (1977).

In scheme 1, the final or penultimate step was an oxidative cyclisation.An alternative approach, using thioacetal coupling, is shown in scheme2. Mercury-mediated unmasking causes cyclisation to the desired compound(Ia).

The thioacetal intermediates may be prepared as shown in scheme 2: thethioacetal protected C-ring [prepared via a literature method: Langley,D. R. & Thurston, D. E., J. Organic Chemistry, 52, 91-97 (1987)] iscoupled to the o-nitrobenzoic acid (or, after hydrolysis, the alkylester) G using a literature procedure. The resulting nitro compoundcannot be reduced by hydrogenation, because of the thioacetal group, sothe tin (II) chloride method is used to afford the amine. This is thenN-protected, e.g., by reaction with a chloroformate or acid chloride,such as p-nitrobenzylchloroformate.

Acetal-containing C-rings can be used as an alternative in this type ofroute with deprotection involving other methods including the use ofLewis Acid conditions.

In the above synthesis schemes, the derivatisation of the A-ring isshown as being complete before the compounds are attached to the solidsupport. This is preferred if the substituents are groups such as alkoxyor nitro. On the other hand, substituent groups such as alkyl or alkenylcould be added to the A-ring after the coupling of the compound to thesolid support. This may be achieved by R₆, R₇, or R₉ being easilyreplaceable groups, such as halogen atoms.

An alternative synthesis approach to those detailed above is to protectthe pro N10 position on the component which will form the A-ring, beforejoining the component which will for the C-ring.

Embodiments of the present invention will now be described by way ofexample with reference to the accompanying drawings in which:

FIG. 1 is a reaction scheme for the synthesis of compounds of formula I;

FIG. 2 is a reaction scheme for the synthesis of alternative compoundsof fomula I;

FIGS. 3a and 3 b are a reaction scheme for the synthesis of compounds offormulae V, IV, III, and II;

FIGS. 4a and 4 b are a reaction scheme for the synthesis of furthercompounds of formulae V, IV, III, and II;

FIG. 5 is a reaction scheme for the synthesis of compounds of formulaIII and IV;

FIGS. 6a, 6 b and 6 c are a reaction scheme for the synthesis ofcompounds of formula VI and VII;

FIG. 7 is a reaction scheme for the synthesis of compounds of formulaIII and IV;

FIGS. 8 and 9 are reaction schemes for the synthesis of compounds offormula III and IV;

FIGS. 10a, b and c are a reaction scheme for the synthesis of compoundsof formula X and XI;

FIG. 11 is reaction schemes for the synthesis of compounds of formulaXIII and XIV; and

FIGS. 12 to 15 are reaction schemes for the synthesis of compounds offormula III and IV.

GENERAL METHODS

Melting points (mp) were determined on an Electrothermal 9100 digitalmelting point apparatus and are uncorrected. Infrared (IR) spectra wererecorded using a Perkin-Elmer Spectrum 1000 spectrophotometer. ¹H- and³C-NMR spectra were recorded on a Jeol GSX 270 MHz FT-NMR spectrometeroperating at 20° C.+/−1° C. Chemical shifts are reported in parts permillion (δ) downfield from tetramethylsilane (TMS). Spin multiplicitiesare described as: s (singlet), bs (broad singlet), d (doublet), dd(doublet of doublets), t (triplet), q (quartet), p (pentuplet) or m(multiplet). Mass spectra (MS) were recorded using a Jeol JMS-DX 303 GCMass Spectrometer (EI mode: 70 eV, source 117-147° C.). Accuratemolecular masses (HRMS) were determined by peak matching usingperfluorokerosene (PFK) as an internal mass marker, and FAB mass spectrawere obtained from a glycerol/thioglycerol/trifluoroacetic acid(1:1:0.1) matrix with a source temperature of 180° C. Optical rotationsat the Na-D line were obtained at ambient temperature using an ADP 220Automatic Polarimeter (Bellingham & Stanley). Flash chromatography wasperformed using Aldrich flash chromatography “Silica Gel-60” (E. Merck,230-400 mesh). Thin-layer chromatography (TLC) was performed using GF₂₅₄silica gel (with fluorescent indicator) on glass plates. All solventsand reagents, unless otherwise stated, were supplied by the AldrichChemical Company Ltd. and were used as supplied without furtherpurification. Anhydrous solvents were prepared by distillation under adry nitrogen atmosphere in the presence of an appropriate drying agent,and were stored over 4 Å molecular sieves or sodium wire. Petroleumether refers to the fraction boiling at 40-60° C.

EXAMPLE 1 Synthesis of PBDs of Formula I (FIG. 1) Overall Synthesis

The compounds with an acid-terminating side chain, 7a-c (R₁₀=Nvoc, Fmoc,Teoc), were prepared by palladium-mediated de-esterification of theappropriate allyl esters. The esters were in turn prepared by Swernoxidation (oxidation of the primary alcohol to an aldehyde whichprovokes spontaneous B-ring closure) of the Nvoc, Fmoc and Teocprotected amino alcohols. The carbamate-protected amino alcohols wereprepared by treating the common amino alcohol intermediate 4 with theappropriate chloroformate in the presence of pyridine. The amino alcoholwas obtained by reduction of the nitro compound 3, which in turn wasassembled by coupling pyrrolidine methanol to the o-nitrobenzoic acid 2.Compound 2 was prepared by selective esterification of the diacid 1 atthe aliphatic acid. Finally, the diacid was obtained by simultaneousnitration and oxidation of the known hydroxypropyloxy vanillic acidderivative 0.

The Troc-protected compound 7d was prepared by an alternative syntheticstrategy involving the use of acetals. The ring closed allyl ester 6dwas prepared by unmasking the acetal protected aldehyde in the presenceof the Troc protected amine. The Troc protected amine was obtainedthrough exposure of the free amine 9 to Troc-C1 in the presence ofpyridine. Reduction with tin chloride furnished the amine 9 from thenitro acetal 8, which in turn was obtained by coupling 2 to theappropriate acetal-protected prolinal.

Allyl Amino Alcohol Intermediate (4)3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (1)

The alcohol 0 (50 g, 0.22 mol) was added portionwise over 1 hour tonitric acid (70%, 400 ml) cooled to 0° C. Once addition was complete,the solution was stirred at 0° C. for 1 hour, then allowed to warm toRT. The semisolid formed was collected by filtration and washed with aminimum of ice/water. The resulting pale yellow solid was redissolved inEtOAc, the solution dried (MgSO₄) and then concentrated to afford thediacid 1 (31 g, 49%). ¹H NMR (270 MHZ): δ2.83-2.79 (t, J=6, 12.5 HZ,2H), 3.94 (s, 3H), 4.37-4.33 (t, J=6, 12.5 MHZ, 2H), 7.18 (s, 1H), 7.46(s, 1H), 10.38 (br.s, 2H).

2-Propene 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoate (2)

A mixture of 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid 1 (20g, 74.3 mmol) and p-toluene sulphonic acid monohydrate (2.3 g, 7.4 mmol)in allyl alcohol (240 mL, 3.5 mol) was refluxed for 7 hours then allowedto cool. The allyl alcohol was then removed in vacuo, and the residuetriturated with dilute HCl acid (3×75 ml) and collected by filtration.This solid was taken up in EtOAc, and the resulting solution washed withwater (3×50 ml) and brine (3×50 ml) and dried over sodium sulphate.Evaporation in vacuo afforded 2 as a white solid (19.27 g, 84%): mp128-130° C.; ¹H-NMR (270 MHZ, CDCl₃) δ2.92 (t, 2H, J=6.35 Hz); 3.94 (s,3H); 4.38 (t, 2H, J=6.41 Hz); 4.65 (d, 2H, J=5.61 Hz); 5.27 (dd, 1H,J₁=1.28 Hz, J₂=19.42 Hz); 5.33 (dd, 1H, J₁=1.28 Hz, J₂=17.04 Hz); 5.92(m, 1H); 7.15 (s, 1H); 7.45 (s, 1H); ¹³C NMR (67.8 MHZ, CDCl₃): δ34.1,56.5, 65.0, 65.4, 108.5, 111.3, 118.3, 122.9, 131.8, 141.1, 149.1,152.6, 167.1, 170.0; IR (Nujol); ν 1730, 1630, 1550, 1430, 1390, 1290,1230, 1190, 1170, 1070, 1030, 1010 cm⁻¹; MS (EI) m/z (relativeintensity): 325 (M^(+•), 19), 251 (3), 213 (2), 196 (3), 211 (3), 113(19), 91 (4), 71 (9), 55 (6); HRMS: calcd. for C₁₄H₁₅NO₈ 325.0798, found232.0773.

2-Propene3-(4-[2′-hydroxymethylpyrrolidinecarboxy]-2-methoxy-5-nitrophenoxy)propanoate(3)

Oxalyl chloride (2.7 ml, 31.0 mmol) was added dropwise to a suspensionof the nitro acid 2 (9 g, 28.0 mmol) and DMF (0.05 ml) in CH₂Cl₂ (150ml), followed by stirring at room temperature for 16 hours The resultingsolution was added dropwise to a stirred solution of pyrrolidinemethanol (3 ml, 31.0 mmol) and triethylamine (8.5 ml, 61.0 mmol) inCH₂Cl₂ (80 ml) at −20° C. (liquid N₂/acetone) followed by stirring atroom temperature for 16 hours under N₂. After quenching with aqueous HCl(1.0 N, 50 ml), the separated organic phase was washed with H₂O (3×25ml) and brine (3×10 ml), dried over magnesium sulphate and evaporated invacuo to afford a crude orange oil. Purification by flash columnchromatography (5% MeOH/EtOAc) afforded 3 as a pale yellow oil (7.9 g,70%): ¹H NMR (270 MHZ, CDCl₃): δ2.22-1.71 (m, 6H); 2.94 (t, J=6.4 Hz,2H); 3.15 (d×d, J=6.5 Hz, 2H); 3.92-3.76 (m, 1H); 3.96 (s, 3H); 4.4 (t,J=6.2 Hz, 2H); 4.67-4.64 (m, 2H); 5.39-5.23 (m, 2H); 6.0-5.86 (m, 1H);6.81 (s, 1H); 7.75 (s, 1H); ¹³C NMR (67.8 MHz, CDCl₃): δ24.4, 28.5,34.1, 49.5, 56.7, 61.6, 64.9, 65.6, 108.9, 109.3, 118.6, 128.2, 131.8,148.2, 154.9, 170.1; IR (film): ν 3394, 2947, 2882, 1735, 1689, 1618,1577, 1521, 1454, 1431, 1386, 1334, 1276, 1221, 1178, 1059, 1002 cm⁻¹;MS (EI) M/Z (relative intensity): 408 (M⁺, 1), 390(4), 377(20), 308(86),296(3), 278(9), 265(35), 252(3), 118(74), 111(3), 108(8), 98(4), 83(14).HRMS: calcd. for C₁₉H₂₅O₆N₂ 377.416, found 377.1711

2-Propene3-(5-amino-4-[2′-hydroxymethylpyrrolidinecarboxy]-2-methoxyphenoxy)propanoate(4)

Solid SnCl₂.2H₂O (21.3 g, 0.095 mol) was added to a stirred solution ofthe nitro alcohol 3 (7.7 g, 0.02 mol) in MeOH (100 ml), and the mixtureheated at reflux for 45 min. The solvent was then evaporated in vacuo,and the residual oil partitioned between EtOAc (50 ml) and aqueoussaturated NaHCO₃ (50 ml) followed by vigorous stirring for 16 hours toaid separation. The combined layers were filtered through Celite andwashed with EtOAc (25 ml). The layers were separated and the resultingorganic phase was washed with H₂O (3×25 ml) and brine (3×10 ml) and thendried over magnesium sulphate. Evaporation in vacuo afforded the amine 4as a dark orange oil (5.6 g, 78%): ¹H NMR (270 MHZ, CDCl₃) δ2.17-1.65(m, 6H); 2.9 (t, J=6.6 Hz, 2H); 3.72-3.46 (m, 3H); 3.75 (s, 3H); 4.2 (t,J=6.8 Hz, 2H); 4.4 (br. d×d, J=9.7 Hz, 2H); 4.65-4.62 (m, 2H); 5.37-5.22(m, 2H); 6.0-5.85 (m, 1H); 6.3 (s, 1H); 6.76 (s, 1H). ¹³C NMR (67.8 MHZ,CDCl₃): δ24.9, 28.7, 34.3, 57.3, 61.2, 64.2, 65.5, 67.4, 102.6, 113.5,118.5, 131.9, 141.1, 150.9, 170.5. IR (film): ν 3354, 2940, 2880, 1734,1621, 1589, 1514, 1453, 1429, 1407, 1265, 1230, 1173, 1110, 1023. MS(EI)M/Z (relative intensity): 378 (M⁺, 60), 278(100), 266(5), 252(9),238(3), 220(4), 206(6), 194(4), 178(3), 166(40), 150(5), 137(20),123(4), 113(4), 107(4), 100(8), 94(12), 84(9). HRMS: calcd. forC₁₉H₂₆O₆N₂ 378.424, found 378.1760.

Example 1(a) Nvoc-PBD Acid (7a) Nvoc Chloroformate

4,5-Dimethoxynitrobenzyl alcohol (2 g, 9.4 mmol) and triphosgene (0.93g, 3.13 mmol) were dissolved in CH₂Cl₂ (50 ml), and the resulting redsuspension stirred vigorously and cooled to 0° C. Pyridine (260 μl, 3.13mmol) was added dropwise, and the resulting green solution stirred atroom temperature for 16 hours to afford a solution of the chloroformatethat was used directly in the next step.

Allyl Nvoc Alcohol (5a)

A solution of freshly prepared (see above) Nvoc chloroformate (9.4 mmol)and pyridine (1.3 ml, 9.5 mmol) was added dropwise to a stirred solutionof the amino alcohol 4 (3 g, 7.9 mmol) in CH₂Cl₂ (60 ml) at 0° C. Themixture was allowed to return to room temperature and stirring continuedfor 3 hours. Evaporation in vacuo afforded an oil which was redissolvedin CH₂Cl₂ (50 ml). The resulting solution was washed with HCl (1.0 N,3×25 ml), H₂O (3×25 ml) and brine (3×10 ml), dried over magnesiumsulphate and evaporated in vacuo to give a dark yellow foam. This waspurified by flash column chromatography (EtOAc) to afford the carbamate5a as a pale yellow foam (3.7 g, 76%): ¹H NMR (270 MHZ, CDCl₃): δ1.6-2.2(m, 6H); 2.9 (t, J=6.2, 2H); 3.4-3.9 (m, 3H); 3.81 (s, 3H); 3.97 and4.01 (2×s, 6H); 4.3 (t, J=6.4 Hz, 2H); 4.63 (d, J=5.9 Hz, 2H); 5.22-5.36(m, 2H); 5.5-5.67 (m, 2H); 5.85-6.0 (m, 1H); 6.84 (s, 1H); 7.09 (s, 1H);7.74 (s, 1H); 8.93 (br. s, 1H). ¹³C NMR (67.8 MHz, CDCl₃): δ25.1, 28.3,34.3, 51.5, 56.4, 56.6, 56.8, 61.0, 63.8, 64.4, 65.4, 66.4, 106.3,108.2, 110.0, 111.9, 118.5, 127.8, 131.5, 131.9, 139.6, 144.4, 148.1,150.3, 153.2, 153.7, 170.3, 170.7. IR (reflectance): ν 3329, 3110, 2937,1728, 1581, 1523, 1453, 1323, 1270, 1175, 1129, 1070, 1031, 1011.MS(FAB) M/Z (relative intensity): 618 (M⁺+1(3)), 473(2), 439(1), 405(1),378(3), 304(7), 278(16), 196(100), 166(28), 151(15), 102(27), 70(9).

Allyl Nvoc PBD (6a)

A solution of DMSO (1.45 ml, 0.02 mol) in CH₂Cl₂ (40 ml) was added over45 minutes to a stirring solution of oxalyl chloride (5.1 ml, 0.01 mol)in CH₂Cl₂ (20 ml) cooled to −40° C. (liquid N₂/chlorobenzene). Stirringwas continued for a further 15 minutes at −40° C., and then a solutionof the NVOC alcohol 5a (3.5 g, 5.7 mmol) in CH₂Cl₂ (45 ml) was addeddropwise over 1 hour. Stirring was continued at −40° C. for a further 45min, and then a solution of Et₃N (3.4 ml, 0.024 mol) in CH₂Cl₂ (20 ml)was added dropwise over 30 minutes and stirring continued for 1 hour.The mixture was then allowed to warm to room temperature before dilutingwith CH₂Cl₂ (20 ml). The organic phase was washed with HCl (1.0 N) (3×50ml), H₂O (3×50 ml) and brine (3×25 ml), dried over magnesium sulphateand then evaporated in vacuo to give a yellow foam. This was purified byflash column chromatography (1% MeOH/CHCl₃) to afford 6a as a paleyellow foam (3.2 g, 91%): ¹H NMR (270 MHZ, CDCl₃): δ1.9-2.2 (m, 6H);2.86 (t, J=6.9 Hz, 2H); 3.45-3.6 (m, 3H); 3.81 (s, 3H); 3.88 and 3.91(2×s, 6H); 4.2-4.4 (m, 3H); 4.61 (m, 2H); 5.19-5.35 (m, 2H); 5.49 (s,2H); 5.7 (br d, J=9.9 Hz, 1H); 5. 82-5.97 (m, 1H); 6.51 (s, 1H); 6.86(s, 1H); 7.25 (s, 1H); 7.66 (s, 1H); ¹³C NMR (67.8 MHZ, CDCl₃): δ23.1,28.7, 30.6, 34.1, 46.5, 56.2, 60.1, 64.6, 65.3, 65.5, 86.1, 107.9,109.2, 110.8, 114.3, 118.4, 126.7, 127.0, 128.1, 131.8, 138.9, 147.9,148.9, 149.9, 153.8, 155.4, 166.8, 170.3. IR (reflectance): ν 3329,3084, 2940, 1713, 1633, 1519, 1454, 1276, 1105, 1067. MS (EI) M/Z(relative intensity) 615 (M⁺, 12), 503(100), 358(4), 261(3), 246(37),231(4), 196(32), 180(24), 166(4), 150(4), 136(6), 70(31).

Acid Nvoc PBD (7a)

Tetrakis(triphenylphosphine) palladium (0.583 g, 0.504 mmol) andmorpholine (4.4 ml, 50.4 mmol) were added to a solution of the allylcarbinolamine 6a (3.1 g, 5.04 mmol) in THF (30 ml) and the mixturestirred for 16 hours. After evaporation in vacuo, the resulting oil wasredissolved in CH₂Cl₂, and the solution washed with HCl (1.0 N) (3×25ml), H₂O (3×25 ml) and brine (3×10 ml), dried over magnesium sulphateand then evaporated in vacuo to give an orange foam. This was purifiedby flash column chromatography (1% MeOH/CHCl₃) to afford 7a as a paleyellow foam (2.25 g, 78%): ¹H NMR (270 MHZ, CDCl₃): δ1.9-2.3 (m, 4H);2.85 (t, J=6.7 Hz, 2H); 3.4-3.6 (m, 3H); 3.68 (s, 3H); 3.87 and 3.9(2×s, 6H); 4.2-4.4 (m, 3H); 5.4-5.48 (m, 2H); 5.7 (br d, J=9.9 Hz, 1H);6.5 (s, 1H); 6.89 (s, 1H); 7.26 (s, 1H); 7.64 (s, 1H); ¹³C NMR (67.8MHZ, CDCl₃): δ23.0, 28.5, 33.9, 46.6, 56.2, 60.4, 64.6, 65.4, 86.1,107.9, 109.3, 110.8, 114.7, 126.5, 126.9, 128.2, 138.9, 147.9, 148.9,149.9, 153.8, 155.5, 167.1, 174.5. IR (reflectance): ν 2939, 2252, 1712,1599, 1522, 1459, 1277, 1221, 1137, 1105, 1066, MS (FAB) M/Z (relativeintensity): 576 (M³⁰+1, 15), 514(3), 381(3), 363(3), 336(3), 319(9),303(2), 293(3), 289(2), 279(4), 266(6), 264(14), 253(3), 245(4),238(10), 215(4), 206(4), 196(100), 192(16), 180(23), 166(32), 151(18),136(10), 123(7), 117(14), 93(28), 73(15), 70(20).

Example 1(b) Fmoc-PBD Acid (7b) Allyl Fmoc Alcohol (5b)

9-Fluorenylmethyl chloroformate (5.65 g, 0.022 mol) was addedportionwise to a stirred solution of the amino alcohol 4 (7.5 g, 0.02mol) and Na₂CO₃ (5.26 g, 0.05 mol) in a mixture of THF (150 ml) andwater H₂O (150 ml) at 0° C. The reaction mixture was allowed to returnto room temperature, stirred for a further 2 h, and then extracted withEtOAc (3×50 ml). The combined organic phase was washed with H₂O (3×50ml) and brine (3×25 ml), dried over magnesium sulphate and evaporated invacuo to give a dark red oil. This was purified by flash columnchromatography (petroleum ether 40-60/EtOAc, 1:1) to afford 5b as a paleyellow oil (8.11 g, 68%): ¹H NMR (270 MHZ, CDCl₃): δ1.72-2.18 (m, 6H);2.9 (t, J=6.4, 2H); 3.43-3.91 (m, 3H); 3.81 (s, 3H); 4.25-4.54 (m, 5H);4.61-4.65 (m, 2H); 5.21-5.37 (m, 2H); 5.85-6.0 (m, 1H); 6.85 (s, 1H);7.31-7.79 (m, 9H); 8.77 (br s, 1H); ¹³C NMR (67.8 MHZ, CDCl₃): δ25.1,28.4, 35.3, 47.0, 56.7, 60.9, 64.3, 65.4, 66.3, 67.1, 106.4, 111.7,118.3, 120.0, 125.2, 127.1, 127.2, 127.8, 131.5, 131.9, 141.3, 143.7,144.4, 150.2, 153.8, 170.3; MS (FAB): 601 (M^(+•)1); HRMS: calcd forC₃₄H₃₆N₂O₈ 600.667, found 600.2175. IR (film): ν 3315, 2952, 1727, 1597,1522, 1452, 1392, 1322, 1174, 1117, 1017.

Fmoc Allyl Carbinolamine Cyclised (6b)

A solution of DMSO (3.4 ml, 0.048 mol) in CH₂Cl₂ (100 ml) was addeddropwise over 45 minutes to a stirred solution of oxalyl chloride (12ml, 0.024 mol) in CH₂Cl₂ (50 ml) at −40° C. (liquid N₂/chlorobenzene).The mixture was stirred at −40° C. for a further 15 minutes and then asolution of the FMOC alcohol 5b (8 g, 0.013 mol) in CH₂Cl₂ (135 ml) wasadded over 1 hour. After stirring for a further 45 minutes at −40° C., asolution of DIPEA (10 ml, 0.057 mol) in CH₂Cl₂ (55 ml) was added over 30minutes and stirring continued for 1 hour. The solution was then allowedto warm to room temperature, diluted with CH₂Cl₂ (100 ml), and theorganic phase washed with HCl (1.0 N) (3×50 ml), H₂O (3×50 ml) and brine(3×25 ml), dried over magnesium sulphate and evaporated in vacuo toafford 6b as a pale cream foam (6.4 g, 80%): ¹H NMR (270 MHZ, CDCl₃):δ1.99-2.1 (m, 4H); 2.83-2.87 (m, 2H); 3.51-3.6 (m, 2H); 3.6-3.8 (m, 1H);3.95 (s, 3H); 4.0-4.58 (m, 7H); 5.17-5.31 (m, 2H); 5.68 (d, J=9.7 Hz,1H); 5.84-5.87 (m, 1H); 6.75 (s, 1H); 7.02-7.75 (m, 9H); ¹³C NMR (67.8MHZ, CDCl₃) δ23.0, 28.7, 34.2, 46.5, 53.5, 56.2, 59.5, 60.1, 64.4, 65.4,68.4, 86.0, 111.2, 114.7, 118.4, 119.9, 124.9, 125.4, 126.8, 127.1,127.8, 128.3, 131.8, 131.9, 141.1, 141.2, 143.1, 143.5, 148.9, 149.9,156.1, 166.9, 170.3; MS (FAB) 599 (M^(+•)+1). IR (reflectance): ν 3318,2950, 1713, 1603, 1517, 1386, 1290, 1177, 1037.

Fmoc Acid Carbinolamine (7b)

Phenylsilane (2.5 ml, 0.02 mol) and tetrakis (triphenylphosphine)palladium(0.232 g, 0.2 mmol) were added to a solution of the FMOCcarbinolamine 6b (6 g, 0.01 mol) in CH₂Cl₂ (80 ml) followed by stirringat room temperature for 16 hours. The reaction was quenched with H₂O (50ml) and extracted with CH₂Cl₂ (3×30 ml). The combined organic phase waswashed with water (3×30 ml), brine (3×25 ml), dried over magnesiumsulphate and evaporated in vacuo to give a dark brown foam. This waspurified by flash column chromatography (MeOH/CHCl₃, 1:99) to afford 7bas a pale beige foam (4.3 g, 77%): ¹H NMR (270 MHZ, CDCl₃): δ1.9-2.2 (m,4H); 2.65-2.85 (m, 2H); 3.4-3.6 (m, 2H); 3.6-3.8 (m, 1H); 3.91 (s, 3H);4.0-4.25 (m, 4H); 4.45-4.5 (m, 1H); 5.68 (d, J=9.5 Hz, 1H); 6.75 (s,1H); 6.9-7.7 (m, 9H); ¹³C NMR (67.8 MHZ, CDCl₃) δ23.0, 28.6, 33.9, 46.5,56.2, 60.3, 64.6, 68.5, 86.0, 111.2, 115.2, 119.8, 124.9, 126.8, 127.1,127.7, 128.2, 140.9, 141.1, 142.9, 143.4, 149.1, 149.9, 156.3, 167.0,174.6. IR (reflectance); ν 3316, 2955, 2609, 2249, 1713, 1601, 1514,1453, 1279, 1036. MS (FAB) M/Z (relative intensity) 560 (M⁺+1).

Example 1(c) Teoc-PBD Acid (7c) Allyl Teoc Alcohol (5c)

Pyridine (0.165 ml, 2.04 mmol) was added dropwise to a solution oftriphosgene (0.605 g, 2.04 mmol) and 2-trimethylsilyl ethanol (1.082 g,9.15 mol) in anhydrous CH₂Cl₂ (100 ml), and the mixture allowed to stirat room temperature for 16 hours. This solution was added dropwise to astirred solution of the amino alcohol 4 (2.30 g, 6.10 mmol) and pyridine(0.987 mL, 0.0122 mol) in anhydrous CH₂Cl₂ (50 mL) at 0° C. (ice bath)under a nitrogen atmosphere. After reaction was complete as indicated byTLC (petroleum ether/ethyl acetate, 1:1), the mixture was washed withcopper (II) sulphate (2×100 mL) and brine (100 mL), dried (MgSO₄) andevaporated in vacuo to give a brown oil. This was purified by flashcolumn chromatography (chlororform/methanol, 99:1) to afford 5c as abrown solid (2.5 g, 78%): ¹H NMR (270 MHz, CDCl₃): δ−0.06 (s, 9H), 1.01(m, 2H), 1.82-2.30 (m, 4H), 2.86 (m, 2H), 3.40-3.75 (m, 7H), 4.15-4.31(m, 4H), 4.6 (m, 2H), 5.15-5.31 (m, 2H), 5.80-5.94 (m, 1H), 6.76 (s,1H), 7.76 (s, 1H), 8.52 (s, 1H); ¹³C NMR (67.8 MHz, CDCl₃) δ−1.47, 17.7,25.1, 28.4, 34.3, 51.6, 56.8, 61.2, 63.5, 64.3, 65.4, 66.8, 106.2,112.1, 113.8, 118.3, 132.0, 132.2, 144.0, 154.1, 170.3; MS (EI): 522(M⁺, 12.8), 435 (7), 350 (13), 319 (77), 262 (27), 206 (13), 149 (88),83 (32), 70 (100); HRMS: Calcd 522.2397, found 522.2351.

Allyl Teoc Carbinolamine PBD (6c)

A solution of DMSO (1.02 mL, 0.014 mol) in dry CH₂Cl₂ (30 mL) was addedto a solution of oxalyl chloride (3.59 mL, 7.185 mmol) in CH₂Cl₂ (25 mL)at −43° C. (chlorobenzene/liq. N₂) under a nitrogen atmosphere. Afterstirring at −43° C. for 45 min, a solution of the TEOC alcohol 5c (2.50g, 4.79 mmol) in dry CH₂Cl₂ (30 mL) was added dropwise to the reactionmixture and stirring continued at −43° C. for a further 45 min. Asolution of triethylamine (3.34 mL, 0.024 mol) in dry DCM (25 mL) wasthen added dropwise, and the vessel allowed to warm to 0° C. Thereaction mixture was diluted with CH₂Cl₂ (150 mL), washed with 1N HCl(100 mL), water (100 mL) and brine (100 mL), dried (MgSO₄) and thenevaporated in vacuo to give crude 6c. This was purified by flash columnchromatography (silica gel, chloroform) to afford 6c as a yellow oil(1.72 g, 69%): ¹H NMR (270 MHz, CDCl₃): δ−0.08 (s, 9H), 0.92 (m, 2H),2.04-2.33 (m, 4H), 3.14 (m, 2H), 3.50-3.75 (m, 4H), 3.93 (s, 3H),4.00-4.40 (m, 4H), 4.67 (m, 2H), 5.26-5.40 (m, 2H), 5.65 (d, 1H, J=9.52Hz), 5.89-5.99 (m, 1H), 6.72 (bs, 1H), 7.23 (s, 1H); ¹³C NMR (67.8 MHz,CDCl₃) δ−1.47, 17.6, 23.0, 28.7, 34.2, 46.4, 56.2, 59.9, 64.3, 65.4,65.5, 85.9, 111.0, 114.7, 118.3, 126.4, 131.8, 132.0, 148.7, 149.7,154.2, 170.0, 170.4; MS (FAB): 629 (0.8), 593 (0.91), 536 (1.5), 493(4.6), 465 (1.0), 449 (1.7), 431 (6.8), 394 (8.1), 368 (1.3), 338 (1.5),304 (5.8), 264 (3.6), 238 (2.2), 204 (1.6), 192 (9.1), 166 (2.5), 149(6.8), 98 (4.3), 73 (100).

Acid Teoc PBD Carbinolamine (7c)

Tetrakis(triphenylphosphine)palladium(0) (190 mg, 0.165 mmol) was addedto a solution of the Teoc-protected carbinolamine 6c (1.72 g, 3.30 mmol)in ethanol (50 mL), and the mixture heated at reflux for 60 minutesafter which time TLC (AcOH/MeOH/chloroform, 1:10:100) indicated thatreaction was complete. The reaction mixture was allowed to cool and wasthen filtered through Celite. Evaporation of the solvent in vacuoafforded 7c as a yellow solid (1.08 g, 68%): ¹H NMR (270 MHz, CDCl₃):δ−0.06 (s, 9H), 0.86 (m, 2H), 1.98-2.20 (m, 4H), 2.8-3.0 (m, 2H),3.40-3.70 (m, 3H), 3.75 (s, 3H), 4.00-4.40 (m, 2H), 5.65 (d, J=8.63 Hz,1H), 6.78 (bs, 1H), 7.21 (s, 1H): ¹³C NMR (67.8 MHz, CDCl₃) δ, −1.5,18.3 , 23.1, 28.7, 34.5, 46.4, 56.1, 58.4, 64.8, 64.9, 85.9, 110.8,115.0, 126.3, 128.7, 148.6, 149.6, 167.2.

Example 1(d) Synthesis of Troc-PBD Acid 7d Prop-2-enyl4-(N-2S-Diethylthiomethylpyrrolidinecarboxy)-2-methoxy-5-nitrophenyl)propanoate(8)

2-Propene 3-(4-carboxy-2-methoxy-5-nitrophenyloxy)propanoate 2: 5 g,15.34 mmol), oxalyl chloride (2 mL, 23 mmol) and 5 drops of DMF werestirred in dry THF (100 mL) for 18 hours. The solvent was then removedin vacuo and the residue dissolved in dry THF (50 mL). This was addeddropwise to a vigorously stirred mixture of(2S)-pyrrolidine-2-carboxaldehyde diethyl thioacetal (3.15 g, 15.34mmol) and triethylamine (1.86 g, 18.41 mmol). The stirring was continuedfor 18 hours. The solvent was then removed in vacuo and the productpurified by flash column chromatography (ethyl acetate) to give 8 (7.48g, 95%) as a yellow oil. ¹H NMR (270 MHZ, CDCl₃): δ7.74 (s, 1H, OCCHC),6.83 (s, 1H, MeOCCHC), 5.98-5.86 (m, 1H, CH₂CHCH₂, 5.33 (d, 1H, J=26.56Hz, OCH₂CHCH₂), 5.28 (d, 1H, J=20.24 Hz, OCH₂CHCH₂), 4.88 (d, 1H, J=3.85Hz, NCHCH), 4.74-4.65 (m, 2H, OCH₂CHCH₂) 4.42 (t, 2H, J=7.69 Hz,CH₂CH₂OC), 3.94 (s, 3H, OCH₃), 3.29-3.21 (m, 2H, NCH₂), 2.96 (p, 2H,J=3.12 Hz, CH₂CH₂O), 2.87-2.67 (m, 4H, SCH₂CH₃), 2.32-1.78 (m, 4H,NCH₂CH₂CH₂)1.38-1.31 (m, 6H, SCH₂CH₃). ¹³C-NMR (CDCl₃): δ15.00, 15.13(SCH₂CH₃), 24.63 (NCH₂CH₂CH₂), 26.28, 26.59, 27.22 (NCH₂CH₂CH₂), 34.13(CH₂CH₂O), 50.19 (NCH₂), 52.80 (NCHCH), 56.60 (OCH₃), 61.08 (NCH), 65.13(CH₂CH₂O), 65.64 (OCH₂CHCH₂), 108.70 (arom. CH), 109.47 (arom. CH),118.55 (OCH₂CHCH₂), 128.58 (CCON), 131.73 (OCH₂CHCH₂), 137.17 (CNO₂),147.98 (CH₂CH₂OC), 154.57 (COCH₃), 166.61 (CON), 170.14 (COO). IR(Nujol) ν=3550-2720, 3000, 2630, 2200, 1740, 1640, 1580, 1530, 1340,1280, 1220, 1180, 1050 cm⁻¹. MS (EI): m/e (relative intensity): 527(M^(+•), 1), 377 (10), 310 (12), 309 (72), 308 (94), 268 (20), 142 (4).HRMS calcd. for C₂₄H₃₅O₇N₂S₂=527.1875, found=527.1885.

5-Amino-3-(4-(2-diethylthiomethyl-(2S)-perhydro-1-pyrroloylcarbonyl)-2-methoxyphenyloxy)2-propenylpropanoate(9)

A solution of 8 (7.21 g, 14.05 mmol) and in(II) chloride (15.85 g, 76mmol) was refluxed for 40 minutes in ethyl acetate (100 mL) then allowedto cool. The solvent was then removed in vacuo and the residue wastriturated with saturated bicarbonate solution at 0° C. EtOAc (50 mL)was added and the reaction stirred overnight. The reaction mixture wasthen filtered through Celite and the filter cake washed with ethylacetate. The combined organics were then washed with water and brine,dried with sodium sulphate and the solvent removed in vacuo. The productwas purified using flash column chromatography (5% MeOH/dichloromethane)to give a yellow oil, (5.87 g, 86%). ¹H NMR (270 MHZ, CDCl₃): δ6.82 (s,1H, arom. CH), 6.28 (s, 1H, arom.CH), 5.99-5.85 (m, 1H, OCH₂CHCH₂), 5.31(dd, 1H, J=1.28 Hz, 27.66 Hz, OCH₂CHCH₂), 5.26 (dd, 1H, J=1.28 Hz, 20.70Hz, OCH₂CHCH₂), 4.71-4.62 (m, 5H, including doublet at 4.62, 2H, J=5.49Hz, NH₂+NCHCH, OCH₂CHCH₂), 4.27 (t, 2H, J=6.59 Hz, CH₂CH₂O), 3.92, (m,1H, NCH), 3.74 (s, 3H, OCH₃), 3.66-3.57 (m, 2H, NCH₂) 2.89 (t, 2H, J=6.6Hz, CH₂CH₂O), 2.83-2.64 (m, 4H, SCH₂CH₃), 2.28-1.80 (m, 4H, NCH₂CH₂CH₂),1.25 (m, 6H, SCH₂CH₃);¹³C NMR (CDCl₃) δ14.20 (SCH₂CH₃), 26.55, 27.23(NCH₂CH₂CH₂), 34.27 (CH₂CH₂O), 53.20 (NCHCH), 56.08 (OCH₃), 60.10 (NCH),60.39 (NCH₂), 64.20 (CH₂CH₂O), 64.41 (OCH₂CHCH₂), 102.26 (arom. CH),113.71 (arom. CH), 118.40 (OCH₂CHCH₂), 131.93 (OCH₂CHCH₂), 141.03(CNH₂), 141.74 (CH₂CH₂OC), 154.56 (COCH₃), 169.69 (CON), 170.53 (COO).IR (neat liquid film) 3500-3000, 3460, 3400, 2970, 1740, 1650, 1535,1470, 1345, 1290, 1225, 1190 cm⁻¹; MS (EI): m/e (relative intensity):482 (M^(+•); 4), 347 (2), 278 (31), 137 (1), 70 (3); HRMS calcd. forC₂₃H₃₄O₅N₂S₂=482.1909, found=482.1925.

3-(4-(2-Diethylthiomethyl-(2S)-perhydro-1-pyrrolylcarbonyl)-2-methoxy-5-(2,2,2-trichloroethyloxycarbonylamino)phenyloxy)2-propenylpropanoate(10)

To a solution of 9 (5.67 g, 11.74 mmol) in dichloromethane (200 mL) wasadd ed pyridine (2.02 mL, 23.48 mmol). To this was added dropwise at 0°C. a solution of trichloroethyl chloroformate(1.616 mL, 11.74 mmol). Thesolution was stirred for a further 1 hour at 0° C. The organics werewashed with 1 N HCl (3×100 mL), water (3×100 mL) brine (100 mL), driedover magnesium sulphate and the solvent removed in vacuo to give a brownoil (6.8 g, 88%) ¹H NMR (270 MHZ, CDCl₃): δ9.14 (bs, 1H, NH), 7.88 (bs,1H, CHCNH), 6.93 (s, 1H, MeOCCHC), 5.99-5.86 (m, 1H, OCH₂CHCH₂), 5.31(dt, 1H, J=1.47 Hz, 27.84 Hz OCH₂CHCH₂), 5.25 (dt, 1H, J=1.29 Hz, 21.61Hz, CH₂CHCH₂),4.89-4.77 (m, 4H, including doublet 1H, J=1.28 Hz,CHCHSEt, NH, CH₂-TrOC), 4.62 (d, 2H, J=1.28 Hz, OCH₂CHCH₂), 3.81 (s, 3H,OCH₃), 3.60 (m, 2H, NCH₂), 2.91 (d, 2H, J=6.42 Hz, CH₂CH₂O), 2.84-2.61(m, 4H, SCH₂CH₃), 1.37-1.23 (m, 6H, SCH₂CH₃); ¹³C NMR (CDCl₃): δ170.33(ester CO), 168.50 (CON), 151.94 (OCO), 150.29 (COCH₃), 144.52(COCH₂CH₂), 131.93 (OCH₂CHCH₂), 131.35 (CNH), 118.29 (OCH₂CHCH₂), 112.21(arom. CH), 105.51 (arom. CH), 95.27 (CCl₃), 76.24 (CH₂TrOC), 74.39(CH₂TrOC), 65.42 (CH₂CH₂O), 61.14 (NCH), 56.30 (OCH₃), 53.00 (NCHCHSEt),34.27 (CH₂CH₂O), 27.30, 26.71, 26.43, 25.24 (NCH₂CH₂CH₂), 15.27, 14.87,14.18 (SCH₂CH₃). MS (EI): m/e (relative intensity): 658, 656 (M^(+•),1), 508 (1), 373 (6), 305 (5), 304 (27), 192 (5), 70 (12).

3-(11-Hydroxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-2-propenylpropanoate(6d)

A solution of 10 (6.8 g, 10.34 mmol) in acetonitrile/water (4:1, 200 mL)was treated with calcium carbonate (2.585 g, 25.85 mmol) andmercuric(II) chloride (7.00 g, 25.85 mmol) and the solution was stirredfor 18 hours. The reaction was then filtered through Celite and thefilter pad washed with ethyl acetate. The organics were collected andwashed with water (3×50 mL), brine (100 mL) and dried over magnesiumsulphate. The solvent was removed in vacuo and the resulting product waspurified by flash column chromatography (ethyl acetate) to give theproduct as a yellow oil (3.67 g, 64%) ¹H NMR (270 MHZ, CDCl₃): δ7.25(arom. CH), 6.86 (s, 1H, arom. CH), 6.00-5.85 (m, 1H, CH₂CHCH₂), 5.67(d, 1H, J=9.71 Hz, TrOC-CH₂) 5.37-5.20 (m, 3H, TrOC-CH₂+OCH₂CHCH₂), 4.65(d, 2H, J=5.67 Hz, CH₂CHCH₂O), 4.36-4.22 (m, 3H, CH₂CH₂O+NCHOH), 3.90(s, 3H, OCH₃), 3.72-3.47 (m, 3H, NCH+NCH₂), 2.91 (t, J=6.41 Hz, CH₂CH₂O)2.29-2.00 (m, 4H, NCH₂CH₂CH₂) ¹³C NMR (67.8 MHZ, CDCl₃): δ170.33 (estercarbonyl CO), 166.17 (CON), 154.4 (OCO), 149.88 (COCH₃), 148.93(COCH₂CH₂), 131.86 (CH₂CHCH₂), 127.48 (arom. CN), 126.24 (CCON), 118.42(OCH₂CHCH₂), 114.48 (arom. CH), 110.82 (arom. CH), 95.09 (CCl₃), 86.42(NCHOH), 74.96 (TrOC-CH₂), 65.47 (OCH₂CHCH₂), 64.43 (CH₂CH₂O), 60.13(NCH), 56.14 (OCH₃), 46.44 (NCH₂), 34.26 (CH₂CH₂O), 28.64 (NCH₂CH₂CH₂),MS (EI) m/z (relative intensity):=552 (M⁺10), 550 (10), 374 (2), 368(5), 304 (15), 192 (8), 70 (24), 55(24). HRMS calcd. forC₂₂H₂₅N₂O₈Cl₃=552.0651, found 3 peaks due to chlorine 552.0646, 550.676,554.0617.

3-(11-Hydroxy-5-oxo-7-methoxy-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxypropanoicacid (7d).

A solution of 6d (3.5 g, 6.35 mmol) was dissolved in ethanol (100 mL).To this was added Tetrakis (triphenylphospine) palladium(0) (350 mg;0.303 mmol) and the solution refluxed for 30 minutes until the reactionwas complete by TLC monitoring. The reaction was then allowed to cooland the filtered through Celite. The EtOH was then removed in vacuo togive the crude material as a yellow solid which was used directly in thenext steps. ¹H-NMR (220 MHZ, CDCl₃): δ7.22 (s, 1H, OCCHCN), 7.01 (s, 1H,MeOCCHC), 6.27 (bs, COOH), 5.67 (d, 1H, J=9.5 Hz, TrOC-CH₂), 5.06 (d,1H, J=12.09 Hz, TrOC-CH₂), 4.29-4.11 (m, 2H, CHOH), 3.85 (s, 3H, OCH₃),3.71 (t, 2H, J=6.97 Hz, CH₂CH₂O), 3.51 (m, 1H, NCH), 2.80 (m, 2H, NCH₂),2.12-1.99 (m, 4H, NCH₂CH₂CH₂), 1.21 (t, 2H, J=6.96 Hz, CH₂CH₂O); ¹³C NMR(67.8 MHZ, CDCl₃): δ=174.27 (acid CH), 167.34 (CON), 154.20 (OCO),149.78 (COCH₃), 148.74 (COCH2CH2), 133.79 (arom. CH), 132.16 (arom. CH),128.66 (arom. CN), 125.87 (CCON), 95.06 (CCl₃), 86.53 (NCHCHOH), 74.95(CH₂-TrOC), 60.67 (NCH), 58.24 (CH₂CH₂O), 56.04 (OCH₃), 46.44 (NCH₂),35.24 (NCH₂CH₂CH₂), 28.59 (NCH₂CH₂CH₂), 23.08 (CH₂CH₂O).

Example 1(e) Synthesis of Resin-bound Protected PBD (JGB-285)

DMF (500 μl) was added to amino Tentagel resin (0.056 g, 0.26 mmol/gloading) in an Alltech tube (8 ml) and the resulting suspension shakenfor 30 min. A solution of Fmoc-PBD-acid 7b(66.6 mg, 0.12 mmol), TBTU(0.038 g, 0.012 mmol) and DIPEA (21 μl, 0.012 mmol) in DMF (1 ml) wasadded and shaking continued for 16 hours. The resin was filtered andrinsed with DMF (5 ml), CH₂Cl₂ (5 ml) and MeOH (5 ml). This procedurewas repeated twice to ensure complete reaction and the resin was thendried in vacuo to afford JGB-285.

Example 1(f) Synthesis of Resin-bound Unprotected PBD (JGB-286)

A solution of piperidine in DMF (20%, 500 μl) was added to the resinJGB-285 and the suspension shaken for 16 hours. The resin was filteredand rinsed with DMF (5 ml), CH₂Cl₂ (5 ml) and MeOH (5 ml). Thisprocedure was repeated twice and the resin was dried in vacuo to affordJGB-286.

EXAMPLE 2 Synthesis of 8-aminopropyl PBD of Formula I (See FIG. 2)Overall Synthesis

The compound 19 was prepared by removal of Fmoc from 18 under standardconditions (piperidine/DMF). The Fmoc carbamate was obtained via Swernoxidation of the alcohol 17, which resulted in spontaneous closure ofthe pyrrolobenzodiazepine B-ring. A number of other oxidation methodsshould also prove effective in promoting the oxidation/cyclizationreaction, for example, the Dess Martin reagent, the TPAP/NMO system orPyridine Sulphur trioxide in DMSO. The alcohol 17 was furnished bytreatment of the amino alcohol 16 with Nvoc-Cl in the presence ofpyridine. As before this is a general procedure applicable to anychloroformate, the choice limited only by compatibility with the PBD andFmoc cleavage conditions. The amine group can also be protected withnumerous other carbamate protecting groups, the most useful in thisinstance being Alloc, Teoc and Noc due to their compatibility with Fmoccleavage conditions. It should be noted that Fmoc itself could beemployed for N-10 protection in which case it would obviously benecessary to employ a different protecting group for the aliphaticnitrogen (see below). The amino alcohol was prepared by tin chloridereduction of the nitro alcohol, which in turn was prepared by couplingpyrrolidine methanol to the o-nitrobenzoic acid 14 under standardconditions. The o-nitrobenzoic acid was prepared by Fmoc protection ofthe amino acid 13. Again, it would be possible to substitute Fmoc with anumber of other protecting groups for example Boc, Alloc, Noc etc. dueto their compatibility with the N10 Nvoc group. It should be noted thatif Fmoc was used to protect the aromatic N10 group, Boc, Alloc, Teoc andNvoc could be used to protect the aliphatic nitrogen. The amino acid 13was prepared by hydrolysis of the ester 12, which in turn was obtainedby simultaneous nitration and deprotection of the Boc protected amine11, which was obtained by a Mitsunobu etherification of methyl vanillatewith Boc aminopropanol.

Boc Amino Ester (11)

A solution diethylazidodicarboxylate (3.38 g, 19.4 mmol) in THF (50 ml)was added dropwise to a solution of methylvanillate (3.53 g, 19.4 mmol),N-Boc-propanolamine (3.4 g, 19.4 mmol) and triphenylphosphine (5.09 g,19.4 mmol) in THF (50 ml) at 0° C. The reaction mixture was allowed towarm to room temperature and stir overnight. Excess solvent was removedby rotary evaporation under reduced pressure and the residue trituratedwith toluene. Precipitated triphenylphosphine oxide was removed byvacuum filtration and the filtrate concentrated in vacuo. The residuewas subjected to flash column chromatography (silica gel, petroleumether 40-60/ethyl acetate, 80/20) and removal of excess eluent affordedthe pure product 11 (4.8 g, 73% yield.). ¹H NMR (270 MHZ, CDCl₃) δ7.65(dd, J=8.43, 2.02 Hz, 1H), 7.54 (d, J=2.02 Hz, 1H), 6.86 (d, J=8.43 Hz,1H), 5.55 (bs, 1H), 4.15 (t, J=5.87 Hz, 2H), 3.93 (s, 3H), 3.90 (s, 3H),3.41-3.35 (m, 2H), 2.09-2.00 (m, 2H) and 1.46 (s, 9H). ¹³C NMR (68.7MHZ, CDCl₃) δ166.9, 156.1, 152.1, 148.8, 123.5, 122.8, 112.0, 111.2,79.0, 68.2, 55.9, 52.0, 38.9, 29.2 and 28.5.

Amino Nitro Ester (12)

The Boc-protected amine 11 (10 g) was added portionwise to cold nitricacid (30 ml, 70%, ice bath), the reaction mixture was allowed warm toroom temperature and stir overnight. The reaction mixture was pouredonto crushed ice (100 g) and the resulting aqueous solution reduced tohalf its original volume by rotary evaporation under reduced pressure.The resulting precipitate was collected by vacuum filtration andrecrystallised from absolute ethanol to afford the product as a yellowcrystalline solid 12 (8.9 g, 87%). ¹H NMR (270 MHZ, CDCl₃) δ7.47 (s,1H), 7.08 (s, 1H), 4.24 (t, J=5.86 Hz, 2H) 3.96, (s, 3H), 3.89 (s, 3H),3.24 (t, J=6.78 Hz, 2H) and 2.32-2.23 (m, 2H).

Amino Nitro Acid (13)

A solution of potassium hydroxide (0.5 g, 8.7 mmol) and the nitrobenzoicacid 12 (1 g, 2.9 mmol) in aqueous methanol (H₂O, 10 ml; methanol, 20ml) was allowed to stir at room temperature for 1 hour and then heatedat reflux until TLC (AcOEt, MeOH, TEA, 1:10:100) revealed the completeconsumption of starting material. Excess methanol was removed by rotaryevaporation and the residual solution diluted with water and neutralisedwith 1N HCl. The neutralised aqueous solution was used directly, withoutfurther purification, in the next synthetic step.

Fmoc Nitro Acid (14)

Fluorenylmethyl chloroformate (0.78 g, 3 mmol) was added portionwise tothe aqueous solution from the previous reaction which had been dilutedwith THF (50 ml) and aqueous sodium carbonate (2.15 g, 50 ml water). Thereaction mixture was then allowed to stir overnight. Excess organicsolvent was removed by rotary evaporation under reduced pressure fromthe reaction mixture, the residual aqueous solution was then washed withethyl acetate (3×20 ml) (to remove excess Fmoc-Cl). The aqueous phasewas acidified with conc. HCl and extracted with ethyl acetate (2×50 ml).The organic phase was dried over magnesium sulphate, filtered andevaporated in vacuo to afford the product 14 (1 g, 70% yield). ¹H NMR(270 MHZ, CDCl₃) δ(Rotamers) 8.21 (bs, 2H), 7.73 (d, J=7.14 Hz, 2H),7.59 (d, J=7.33 Hz, 2H) 7.40-7.13 (m, 5H), 6.47 and 5.70 (2×bs, 1H),4.54-3.88 (m, 5H), 3.77 (s, 3H), 3.44-3.42 (m, 2H) and 2.04-1.90 (m,2H). ¹³C NMR (68.7 MHZ, CDCl₃) δ168.7, 156.9, 152.1, 149.8, 143.7,141.9, 141.3, 127.7, 127.0, 124.9, 120.6, 120.0, 111.1, 107.8, 68.5,66.4, 56.4, 47.3, 39.1 and 28.4.

Fmoc Nitro Alcohol (15)

A catalytic amount of DMF (2 drops) was added to a solution of the acid14 (1.16 g, 2.36 mmol) and oxalyl chloride (0.33 g, 2.6 mmol) in drydichloromethane (20 ml) and the reaction mixture was allowed to stirovernight. The resulting acid chloride solution was cooled to 0° C. andtreated dropwise with a solution of pyrrolidinemethanol (0.26 g, 2.57mmol) and triethylamine (0.52 g, 5.14 mmol) in dry dichloromethane (15ml). Thin layer chromatography, performed shortly after the end of theaddition of amine, revealed that reaction had gone to completion. Thereaction mixture was washed with HCl (1N, 1×50 ml) and water (2×20 ml)and dried over magnesium sulphate. Removal of excess solvent affordedthe crude product which was subjected to flash column chromatography(silica gel, gradient elution,1% methanol in chloroform to 2% methanolin chloroform) to afford the required amide 15 (1.1 g, 81%). ¹H NMR (270MHZ, CDCl₃) δ7.75 (d, J=7.33 Hz, 2H), 7.67 (s, 1H), 7.60 (d, J=6.96 Hz,2H), 7.41-7.26 (m, 4H), 6.78 (s, 1H), 5.66 (bs, 1H), 4.48-4.39 (m, 3H),4.23-4.13 (m, 3H), 3.91-3.79 (m, 5H), 3.45-3.42 (m, 2H), 3.18-3.13 (m,2H) and 2.08-1.70 (m, 6H). ¹³C NMR (68.7 MHZ, CDCl₃) δ168.5, 156.5,154.7, 148.2, 143.9, 141.3, 137.0, 128.0, 127.7, 127.0, 124.9, 120,108.9, 108.0, 68.4, 66.2, 66.0, 61.5, 56.6, 53.5, 47.3, 39.0, 28.9, 28.4and 24.4.

Fmoc Amino Alcohol (16)

A solution of the nitroamide 15 (3 g, 5.22 mmol) and SnCl₂ 2H₂O (6.15 g,27.15 mmol) in methanol (60 ml) was heated at reflux for 2 hours. Thereaction mixture was concentrated to ⅓ of its original volume andcarefully treated with saturated aqueous sodium bicarbonate solution(vigorous effervescence!) until pH8 was obtained. The mixture wasallowed to stir vigorously with ethyl acetate (100 ml) overnight andthen filtered through celite to remove precipitated tin salts. Theaqueous phase was extracted with ethyl acetate (50 ml) and the combinedorganic phase was dried over magnesium sulphate. Removal of excesssolvent afforded the desired amine as a dark yellow oil 16 (1.93 g,68%). ¹H NMR (270 MHZ, CDCl₃) δ7.75 (d, J=7.51 Hz, 2H), 7.61 (d, J=7.33Hz, 2H), 7.40-7.26 (m, 4H), 6.72 (s, 1H), 6.25 (s, 1H), 5.95 (bs, 1H),4.43-4.04 (m, 6H), 3.67-3.42 (m, 9H) and 2.11-1.7 (m, 6H). ¹³C NMR (68.7MHZ, CDCl₃) δ171.7, 156.6, 150.8, 144.0, 141.3, 140.6, 127.6, 127.0,125.0, 119.9, 112.0, 102.2, 68.0, 66.6, 66.4, 61.0, 56.6, 51.0, 47.3,39.5, 29.1, 28.5 and 24.9.

Fmoc Nvoc Alcohol (17)

A solution of 4,5-dimethoxy-2-nitrobenzylchloroformate (1.44 g, 5.23mmol) in dichloromethane (40 ml) was added dropwise to a solution of theamine 16 (2.59 g, 4.75 mmol) and pyridine (0.41 g, 5.23 mmol) indichloromethane (60 ml) at 0°C. After 3 hours the reaction mixture waswashed with HCl (1N, 2×100 ml), water (2×100 ml) and brine (1×100 ml).The organic phase was dried over magnesium sulphate and removal ofexcess solvent gave the crude product, which was subjected to flashcolumn chromatography (silica gel, ethyl acetate followed by 1% methanolin ethyl acetate) to afford the pure carbamate 17 (3.2 g, 86%). ¹H NMR(270 MHZ, CDCl₃) δ8.94 (br,s 1H) 7.74 (d, J=7.51 Hz, 2H), 7.71 (s, 1H),7.61 (d J=7.33 Hz, 2H), 7.40-7.25 (m, 4H), 7.08 (s, 1H), 6.80 (s, 1H),5.62 (d, J=15.02 Hz, 1H), 5.50 (d, J=15.02, 1H), 4.44-4.41 (m, 3H),4.24-4.13 (m,3H), 3.99 (s, 3H), 3.94 (s, 3H), 3.70-3.44 (m, 9H), and2.17-1.72 (m, 6H). ¹³C NMR (68.7 MHZ, CDCl₃) δ171.7, 164.0, 156.6,153.7, 153.3, 150.1, 148.1, 144.3, 144.0, 141.3, 139.6, 131.3, 127.6,127.0, 125.0, 119.9, 110.7, 110 1, 108.2, 105.5, 68.1, 66.4, 66.1, 63.9,60.9, 56.6, 56.4, 56.2, 47.3, 39.5, 28.9, 28.3 and 25.1.

Fmoc Nvoc Carbinolamine (18)

A solution of DMSO (0.8 ml, 11.4 mmol) in dry dichloromethane (15 ml)was added dropwise, over 30 minutes, to a solution of oxalyl chloride(0.72 g, 5.71 mmol) in dry dichloromethane (15 ml) at −45° C. under anitrogen atmosphere. The reaction mixture was allowed to stir for 30minutes before the addition of the substrate 17 (3.2 g, 4.08 mmol) indry dichloromethane (35 ml) over 50 minutes whilst maintaining thetemperature of the reaction at −45° C. The reaction mixture was thenallowed to stir at −45° C. for a further 45 minutes. A solution oftriethylamine (2.15 ml, 16.2 mmol) in dry dichloromethane (10 ml) wasadded dropwise over 25 minutes at −45° C. and the reaction mixtureallowed to stir for a further 30 minutes at −45° C. before being allowedto warm to room temperature. The reaction mixture was washed with 1N HCl(1×75 ml), water (1×75 ml), brine (1×75 ml) and dried over magnesiumsulphate. Removal of excess solvent furnished the crude product whichwas subjected to flash column chromatography (silica gel, ethyl acetate)to afford the cyclized product 18 (1.92 g, 60% yield). ¹H NMR (270 MHZ,CDCl₃) δ7.74 (d, J=7.51 Hz, 2H), 7.60-7.59 (m, 3H), 7.40-7.23 (m, 4H),7.22 (s, 1H), 6.83 (s, 1H), 6.50 (s, 1H), 5.88 (bs, 1H), 5.72 (d, J=9.34Hz, 1H), 5.45-5.38 (m, 2H), 4.59 (bs, 1H), 4.42 (d, J=7.14 Hz, 2H),4.22-4.08 (m, 3H), 3.86 (s, 3H), 3.76 (s, 3H), 3.68 (s, 3H), 3.59-3.44(m, 4H) and 2.12-2.02 (m, 6H). ¹³C NMR (68.7 MHZ, CDCl₃) δ166.9, 156.6,155.4, 153.8, 150.1, 148.8, 148.1,144.0, 141.3, 139, 128.2, 127.7,127.0, 126.7,126.5, 125.0, 120.0, 113.7, 110.5, 109.7,108.1, 86.2, 68.4,66.3, 65.4, 60.3, 56.3, 56.2, 56.0, 47.3, 46.5, 39.4, 29.7, 28.7 and23.1.

Amino Nvoc Carbinolamine (19)

The Fmoc protected amine 18 (0.5 g, 0.64 mmol) was added to a solutionof piperidine (1 g, 11.7 mmol) in dichloromethane (10 ml). After 2 hoursTLC revealed the continued presence of starting material DMF was addedas a co-solvent and reaction proceeded to completion over the next 30minutes. The reaction mixture was diluted with ethyl acetate (50 ml),washed with water (25 ml), brine (25 ml) and dried over magnesiumsulphate. Removal of excess solvent afforded a yellow crystallineproduct which was recrystallised from ethyl acetate and petroleum ether40-60 (0.123 g, 34% yield). ¹H NMR (270 MHZ, CDCl₃) δ7.61 (s, 1H), 7.21(s, 1H), 6.90 (s, 1H), 6.50 (s, 1H), 5.70 (d, J=9.70 Hz, 1H), 5.44 (bs,2H), 4.13-4.11 (m, 1H), 3.96-3.40 (m, 13H), and.2.18-1.90 (m, 6H). ¹³CNMR (68.7 MHZ, CDCl₃) δ167.1, 155.1, 153.8, 150.0, 148.7, 147.9, 138.8,128.5, 127.3, 126.5, 114.3, 110.4, 109.5, 107.9, 86.0, 67.1, 65.1, 60.7,56.3, 56.1, 53.3, 38.7, 28.7, 28.0 and 23.1.

Example 3a Synthesis of PBD-Triglycine 30 (FIGS. 3 a & 3 b)

This example was carried out to prove the general method of synthesis.

Resin Deprotection

Fmoc-aminoethyl photolinker NovaSyn TG resin 20 (0.35 g, 0.23 mmol/gloading) was placed in a peptide vessel, fitted with a sinter. After theaddition of 20% piperidine in DMF (3 ml), the vessel was shaken for 3hours. The deprotected resin 21 was then separated by filtration andrinsed with NMP (3 ml), MeOH (3 ml) and CH₂Cl₂ (3 ml). The wholeprocedure was repeated twice before drying the resin in vacuo.

Coupling Conditions

DMF (2 ml) was added to resin 21 and the suspension shaken for 30 min. Asolution of Fmoc-glycine (0.24 g, 0.805 mmol), TBTU (0.26 g, 0.805 mmol)and DIPEA (140 μl, 0.805 mmol) in DMF (2 ml) was added and shakingcontinued for 20 hours. The coupled resin 22 was then filtered andrinsed with NMP (5 ml), MeOH (5 ml) and CH₂Cl₂ (5 ml). The wholeprocedure was repeated once before drying the resin in vacuo. Thecoupling efficiency was monitored by the addition of bromophenol blue inDMF (0.2 ml).

Acetylation (Endcapping) Conditions

Ac₂O (20%) and pyridine (30%) in CH₂Cl₂ (5 ml) was added to resin 22 andthe suspension was shaken for 2 hours. The acetylated resin was filteredand washed with CH₂Cl₂ (5 ml), EtOH (5 ml) and a further aliquot ofCH₂Cl₂ (2 ml). The whole procedure was repeated once before drying theresin in vacuo. The effectiveness of acetylation was monitored by theaddition of bromophenol blue in DMF (0.5 ml).

Deprotection Conditions

Piperidine (20%) in DMF (2 ml) was added to the acetylated resin 22 andthe suspension was shaken for 12 hours. The deprotected resin 23 wascollected by filtration and rinsed with NMP (5 ml), MeOH (5 ml) andCH₂Cl₂ (5 ml). The whole procedure was repeated twice before drying theresin in vacuo.

Addition of Two Further Glycine Units

The previous coupling (21-22), acetylation and deprotection (22-23)steps were repeated twice (23-27) until the resin-bound tripeptide 27was obtained.

Coupling to the PBD Unit (Fmoc-PBD)

Triglycine resin 27 (0.12 g, 0.235 mmol/g loading) was placed in apeptide synthesis vessel fitted with a sinter. DMF (3 ml) was added andthe vessel was shaken for 30 min. A solution of the Fmoc-PBD acid 7b(0.15 g, 0.28 mmol), TBTU (0.09 g, 0.28 mmol) and DIPEA (50 μl, 0.28mmol) in DMF (3 ml) was added, and shaking continued for 20 hours.Bromophenol blue indicator (30 μl) was added to monitor the progress ofthe reaction. The coupled resin 28b was collected by filtration andrinsed with DMF (5 ml), NMP (5 ml) and CH₂Cl₂ (5 ml). The wholeprocedure was repeated twice before drying the resin in vacuo.

Acetylation (Endcapping) Conditions

Ac₂O (20%) and pyridine (30%) in CH₂Cl₂ (5 ml) was added to resin 28band the vessel shaken for 2 hours. The acetylated resin was filtered andwashed with CH₂Cl₂ (5 ml), EtOH (5 ml) and further CH₂Cl₂ (2 ml). Thewhole procedure was repeated once and the resin 29 dried in vacuo. Theeffectiveness of acetylation was monitored by the addition ofbromophenol blue in DMF (0.5 ml).

Deprotection to Free PBD

Piperidine (20%) in DMF (2 ml) was added to the acetylated resin 28a andthe vessel shaken for 12 hours. The resin 29 was collected by filtrationand rinsed with NMP (5 ml), MeOH (5 ml) and CH₂Cl₂ (5 ml). Thisprocedure was repeated twice and the resin dried in vacuo.

Synthesis of Nvoc-PBD Triglycine 28a (FIG. 3b)

Triglycine resin 27 (0.16 g, 0.235 mmol/g loading) was placed in apeptide vessel fitted with a sinter. DMF (3 ml) was added and the vesselshaken for 30 min. A solution of the Nvoc-PBD acid 7a (0.22 g, 0.38mmol), TBTU (0.12 g, 0.38 mmol) and DIPEA (65 μl, 0.38 mmol) in DMF (3ml) was added, and shaking continued for 20 hours. Bromophenol blueindicator (30 μl) was added to monitor the progress of the reaction.Resin 28a was collected by filtration and rinsed with DMF (5 ml), NMP (5ml) and CH₂Cl₂ (5 ml). The whole procedure was repeated twice beforedrying the resin in vacuo.

Acetylation (Endcapping) Conditions

Ac₂O (20%) and pyridine (30%) in Ch₂Cl₂ (5 ml) was added to resin 28aand the vessel shaken for 2 hours. The acetylated resin was collected byfiltration and washed with CH₂Cl₂ (5 ml), EtOH (5 ml) and further CH₂Cl₂(2 ml). The whole procedure was repeated once before drying the resin 29in vacuo. The effectiveness of acetylation was monitored by the additionof bromophenol blue in DMF (0.5 ml).

Photolysis

A suspension of beads bearing the Nvoc-PBD in DMF was simultaneouslyN10-deprotected and cleaved from the resin by irradiating at 365 nm for2 hours (Spectrolinker XL 1000 UV Crosslinker, Spectronics Corporation)to afford a 1 mmol stock solution of 30 which was used directly in theMTT assay (Example 3b).

Example 3(b) General MTT Assay Method

The ability of agents to inhibit the growth of U937 chronic humanhistiocytic leukemia cells or K562 human chronic myeloid leukemia cellsin culture was measured using the MTT assay (Mosmann, 1983). This isbased on the ability of viable cells to reduce a yellow solubletetrazolium salt, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma Chemical Co.), to an insoluble purple formazanprecipitate. Following drug treatment, the cells were transferred to96-well microtitre plates with 10⁴ cells per well and 8 wells persample. The plates were incubated at 37° C. in a humidified atmospherecontaining 5% CO₂. Following incubation of the plates for 4 days (toallow control cells to increase in number by 10-fold), 20 μL of a 5mg/mL solution of MTT in phosphate-buffered saline was added to eachwell and the plates incubated further for 5 hours. The plates were thencentrifuged for 5 minutes at 300 g and the bulk of the medium removedfrom the cell pellet, leaving 10-20 μL per well. DMSO (200 μL) was addedto each well, and the samples agitated to ensure complete mixing. Theoptical density was then read at a wavelength of 550 nm on a TitertekMultiscan ELISA plate reader and the dose-response curve constructed.The IC₅₀ value was read as the dose required to reduce the final opticaldensity to 50% of the control value.

MTT Assay of PBD-Triglycine

The MTT assay was used to evaluate the cytoxicity of the previouslyprepared PBD-Triglycine (compound 30), from Example 3a. The assay foundthe IC₅₀ to be 0.59 μM.

Example 4(a) Synthesis of the Resin-bound Tripeptide Library (FIGS. 4 aand 4 b)

Following Example 3, this procedure was used to synthesize a tripeptidelibrary.

Resin Deprotection

Fmoc-aminoethyl photolinker NovaSyn TG resin 20 (1.35 g, 0.23 mmol/gloading) was weighed into 27 Alltech tubes in 50 mg portions. Piperidine(20%) in DMF (0.5 ml) was added to each tube, and the tubes were thenplaced onto an orbital shaker for 3 hours. The deprotected resin 21 wascollected by filtration using a Supelco Vacuum Manifold and rinsed withDMF (2 ml) and CH₂Cl₂ (2 ml). The whole procedure was repeated twicebefore drying the resin in vacuo.

Coupling Conditions

DMF (0.5 ml) was added to each Alltech tube containing resin 21, and thetubes were shaken for 30 min. Fmoc-glycine (0.306 g, 1.03 mmol) in DMF(3.4 ml) was added to the first 9 tubes; Fmoc-valine (0.36 g, 1.06 mmol)in DMF (3.54 ml) to the next 9 tubes and Fmoc-phenylalanine (0.396 g,1.03 mmol) in DMF (3.4 ml) to the final 9 tubes. TBTU (0.972 g, 3.03mmol) in DMF (10 ml) and DIPEA (540 μl 3.1 mmol) were added to all 27tubes and shaking was continued for 20 hours. The coupled resin 31 wascollected by filtration and rinsed with DMF (5 ml), CH₂Cl₂ (5 ml) andEtOH (5 ml). The whole procedure was repeated twice before drying theresin in vacuo. The coupling efficiency was monitored by the addition ofa few drops of 10% DIPEA/DMF and 1% TNBS/DMF. The resin remainedcolourless when coupling was complete.

Acetylation (Endcapping) Conditions

Ac₂O (20%) and pyridine (30%) in Ch₂Cl₂ (1 ml) were added to eachAlltech tube containing resin 31, and the tubes were shaken for 2 hours.The acetylated resin was filtered and washed with CH₂Cl₂ (5 ml), EtOH (5ml) and further CH₂Cl₂ (2 ml). The whole procedure was repeated oncebefore drying the resin in vacuo.

Deprotection Conditions

Piperidine (20%) in DMF (0.5 ml) was added to each tube of acetylatedresin 31, and the tubes shaken for 12 hours. The deprotected resin 32was collected by filtration and rinsed with DMF (5 ml) and CH₂Cl₂ (5ml). The whole procedure was repeated twice before drying the resin invacuo.

Coupling of Two Further Amino Acid Units

The previous coupling (21-31), acetylation and deprotection (31-32)steps were repeated twice using the appropriate Fmoc-protected AminoAcids in each Alltech tube (32-36) to achieve all possible combinations.This resulted in the resin-bound tripeptide library 36.

Coupling to the Fmoc-PBD Unit (FIG. 4b)

DMF (0.5 ml) was added to each Alltech tube containing resin 36 and thetubes were shaken for 30 min. Fmoc-PBD acid 7b (0.866 g, 1.55 mmol) inDMF (5.2 ml), TBTU (0.486 g, 1.55 mmol) in DMF (5 ml) and DIPEA (270 μl,1.55 mmol) were added and shaking was continued for 20 hours. Thecoupled resin 37 was collected from each tube by filtration and rinsedwith CH₂Cl₂ (5 ml), EtOH (5 ml) and further CH₂Cl₂ (2 ml). The wholeprocedure was repeated twice before drying the batches of resin invacuo.

Acetylation (Endcapping) Conditions

Ac₂O (20%) and pyridine (30%) in CH₂Cl₂ (1 ml) were added to each tubeof resin 37 and the tubes were shaken for 2 hours. Acetylated resin fromeach Alltech tube was collected by filtration and washed with CH₂Cl₂ (5ml), EtOH (5 ml) and further CH₂Cl₂ (2 ml). The whole procedure wasrepeated once before drying the resin in vacuo. The effectiveness ofacetylation was monitored by the addition of a few drops of 10%DIPEA/DMF and 1% TNBS/DMF.

Deprotection Conditions

Piperidine (20%) in DMF (0.5 ml) was added to each tube of acetylatedresin 37 and the tubes were shaken for 12 hours. The batches ofdeprotected resin 38 were collected by filtration and rinsed with DMF (5ml), CH₂Cl₂ (5 ml) and MeOH (5 ml). The whole procedure was repeatedtwice before drying the residue in vacuo to afford batches of resin 38.

Photolysis

A suspension of beads bearing the deprotected resin 38 were cleaved fromthe resin by irradiating at 365 nm for 2 hours (Spectrolinker XL 1000 UVCrosslinker, Spectronics Corporation) to afford a 1 mmol stock solutionwhich was used directly in the MTT assay (EXAMPLE 4b).

Example 4(b) Screening of a 27 Member Combinatorial Library Prepared onBeads

The combinatorial library synthesised in example 4(a) was screened usingthe MTT assay as previously described.

Amino-acid Sequence of Combinatorial Compound Units IC₅₀ (μM) 1 GGG 0.592 GGV 0.63 3 GGF 0.56 4 GVG 0.55 5 GVV 0.52 6 GVF 0.64 7 GFG 0.63 8 GFV0.78 9 GFF 0.63 10 VGG 0.59 11 VGV 0.33 12 VGF 0.58 13 VVG 0.58 14 VVV0.58 15 VVF 0.53 16 VFG 0.46 17 VFV 0.56 18 VFF 0.56 19 FGG 0.54 20 FGV0.54 21 FGF 0.57 22 FVG 0.58 23 FVV 0.54 24 FVF 0.69 25 FFG 0.54 26 FFV0.40 27 FFF 0.59 GW/613 — 0.33 G = GLYCINE V = VALINE F = PHENYLALANINE

These results demonstrate that varying the amino acid sequence affectsthe cytotoxicity of the PBDs.

Example 5(a) Preparation of a 27-Member Tripeptide-PBD Library on Crownsfor Solution Phase Testing

A 27-member library was prepared on Chiron Technology crowns fromFmoc-protected Glycine, Leucine and Phenylalanine building blocks, andan NVOC-protected PBD unit using the Multipin™ Synthesis Kit and thesame general reaction scheme as exemplified in Example 4(a).

Crown Deprotection

Twenty-seven Fmoc-Rink amide O-series crowns (loading: 2.2 μM per crown)were attached to the first twenty-seven pins of a 98-pin block. Theblock was inverted and placed in a vessel containing a solution ofpiperidine (20%) in DMF (50 mL, anhydrous) on a shaker (Heidolph,Titramax 100). After 30 min. the block was removed from the containerand excess piperidine/DMF allowed to drain away. The block was theninverted, placed in a vessel containing fresh DMF (50 mL), and the wholeassembly agitated for 5 min. Finally, the crowns were washed twice for 2minutes with methanol before allowing to air-dry for 20 min.

Preparation of Activated Amino Esters

A solution of N-Fmoc-Glycine (128.4 mg, 0.43 mmol), DIC (55 mg, 0.43mmol) and HOBt (70 mg, 0.52 mmol) in DMF (2160 μL) was agitated for 20min, and aliquots (200 μL) were added to the first nine wells (H1-H9) ofa deep 98-well microtitre plate. Similar solutions of active esters wereprepared from N-Fmoc Leucine (152.7 mg) and N-Fmoc-Phenylalanine (167.4mg), and aliquots (200 μL) were dispensed into wells H10-G6 and G7-F3,respectively (see Table 1 below).

TABLE 1 Distribution of N-Fmoc-protected Amino Acids into Wells H1 toF3. H G F 1 Glycine Leucine Phenylalanine 2 Glycine LeucinePhenylalanine 3 Glycine Leucine Phenylalanine 4 Glycine Leucine 5Glycine Leucine 6 Glycine Leucine 7 Glycine Phenylalanine 8 GlycinePhenylalanine 9 Glycine Phenylalanine 10 Leucine Phenylalanine 11Leucine Phenylalanine 12 Leucine Phenylalanine

Coupling Reaction

Each well was doped with a small amount of bromophenol blue indicator(25 μL of 6.6 mg in 10 mL of DMF) and the crown array immersed in thewells. The crowns (previously colourless) instantly turned blueindicating the presence of free amines. The block/deep well plateassembly was agitated on a shaker for 18 hours after which time all ofthe crowns became virtually colourless indicating that the couplingreactions had gone to completion. The crown array was then removed fromthe microtitre plate, excess coupling reagent allowed to drain, and thecrown array washed once with DMF (50 mL, five min) and twice withmethanol (100 mL, 2 min). Finally, the crown array was allowed to airdry for 20 min.

Deprotection

The crown array was inverted and placed in a vessel containing asolution of piperidine (20%) in DMF (50 mL, anhydrous) on a shaker(Heidolph, Titramax 100). After 30 min, the block was removed from thecontainer and excess piperidine/DMF allowed to drain away. The block wasthen inverted, placed in a vessel containing fresh DMF (50 mL) and thewhole assembly agitated for 5 min. Finally, the crowns were washed twicefor 2 minutes with methanol before allowing to air-dry for 20 min.

Coupling to the Second Amino Acid

The deprotected crown array was immersed in a deep well microtitre platecharged with freshly prepared solutions of the activated esters ofN-Fmoc-Glycine, N-Fmoc-Leucine and Fmoc-Phenylalanine according to thepattern shown in Table 2 below. The block/deep well plate assembly wasagitated on an orbital shaker for 18 hours after which time all of thecrowns had become virtually colourless indicating that the couplingreactions had gone to completion. The crown array was then removed fromthe microtitre plate, excess coupling reagent allowed to drain, and thecrown array washed once with DMF (50 mL, five min) and twice withmethanol (100 mL, 2 min). Finally, the crown array was allowed to airdry for 20 min.

TABLE 2 Distribution of N-Fmoc-Protected Amino Acids into Wells H1 toF3. H G F 1 Glycine Leucine Phenylalanine 2 Glycine LeucinePhenylalanine 3 Glycine Leucine Phenylalanine 4 Leucine Phenylalanine 5Leucine Phenylalanine 6 Leucine Phenylalanine 7 Phenylalanine Glycine 8Phenylalanine Glycine 9 Phenylalanine Glycine 10 Glycine Leucine 11Glycine Leucine 12 Glycine Leucine

Deprotection

The crown array was inverted and placed in a container charged with asolution of piperidine (20%) in DMF (50 mL, anhydrous) on a shaker(Heidolph, Titramax 100). After 30 min, the block was removed from thecontainer and excess piperidine/DMF allowed to drain away. The block wasthen inverted, placed in a vessel containing fresh DMF (50 mL) and thewhole assembly agitated for 5 min. Finally, the crowns were washed twicefor 2 minutes with methanol before allowing to air-dry for 20 min.

Coupling of the Third Amino Acid Unit

The deprotected crown array was immersed in a deep well microtitre platecharged with freshly prepared solutions of the activated esters ofN-Fmoc-Glycine, N-Fmoc-Leucine and Fmoc-Phenylalanine according to thepattern shown in Table 3 below. The block/deep well plate assembly wasagitated on an orbital shaker for 18 hours after which time all of thecrowns had become virtually colourless indicating that the couplingreactions had gone to completion. The crown array was then removed fromthe microtitre plate, excess coupling reagent allowed to drain, and thecrown array washed once with DMF (50 mL, five min) and twice withmethanol (100 mL, 2 min). Finally, the crown array was allowed to airdry for 20 min.

TABLE 3 Distribution of N-Fmoc-protected Amino Acids into Wells H1 toF3. H G F 1 Glycine Glycine Glycine 2 Leucine Leucine Leucine 3Phenylalanine Phenylalanine Phenylalanine 4 Glycine Glycine 5 LeucineLeucine 6 Phenylalanine Phenylalanine 7 Glycine Glycine 8 LeucineLeucine 9 Phenylalanine Phenylalanine 10 Glycine Glycine 11 LeucineLeucine 12 Phenylalanine Phenylalanine

Deprotection

The crown array was inverted and placed in a vessel containing asolution of piperidine (20%) in DMF (50 mL, anhydrous) on a shaker(Heidolph, Titramax 100). After 30 min, the block was removed from thecontainer and excess piperidine/DMF allowed to drain away. The block wasthen inverted, placed in a vessel containing fresh DMF (50 mL) and thewhole assembly agitated for 5 min. Finally, the crowns were washed twicefor 2 minutes with methanol before allowing to air-dry for 20 min.

Attachment of PBD Capping Unit

A solution of Nvoc-PBD acid (745 mg, 1.29 mmol), DIC (16 mg, 1.29 mmol)and HOBt (209 mg, 1.55 mmol) in DMF (6.48 ml) was agitated for 20minutes and then dispensed into all twenty-seven wells. The block/deepwell microtitre plate assembly was agitated on a shaker for 48 hours.The crown array was then removed from the microtitre plate, excesscoupling reagent allowed to drain, and the crown array washed once withDMF (50 mL, five min) and twice with methanol (100 mL, 2 min). Finally,the crown array was allowed to air dry for 20 min.

Cleavage

Prior to cleavage, the crowns were washed successively with DMF,toluene, methanol and dichloromethane to remove any non-covalentcontaminants. The crown array was then immersed in twenty-seven racked(but individual) 1 mL polypropylene tubes each containing TFA/H₂O (300μL, 95:5, v/v), and the block/rack assembly was agitated on an orbitalshaker for 2 hours at room temperature. Excess TFA was removed byparallel evaporation under nitrogen (supplied by a glass manifold with 8outlets) followed by final drying in vacuo over 48 hours to afford thefree N10-Nvoc-Protected PBD-tripeptides.

EXAMPLE 5(b) Photolytic Cleavage and MTT Assay Method

The same assay method as Example 3(b) was used. Cells at a density of5×10⁴ cells/mL were continuously incubated with each member of the27-mer library at a final concentration of 0.3 μM (6.6 μmoles/tube/ml).Aliquots of each of the compounds of the 27-member library were eitherleft without UVA (365 nm) exposure or were exposed to UVA (365 nm) for 2h prior to their addition to the cell suspension. Following addition ofthe compounds, the cells were transferred to 96-well microtitre plates,10⁴ cells per well, 8 wells per sample. Plates were incubated at 37° C.in a humidified atmosphere containing 5% CO₂. Following incubation ofthe plates for 4 days (to allow control cells to increase in number by10-fold), 20 μL of a 5 mg/mL solution of MTT in phosphate-bufferedsaline was added to each well and the plates further incubated for 5hours. The plates were then centrifuged for 5 minutes at 300 g and thebulk of the medium was removed from the cell pellet, leaving 10-20 μLper well. DMSO (200 μL) was added to each well, and the samples agitatedto ensure complete mixing. The optical density was then read at awavelength of 550 nm on a Titertek Multiscan ELISA plate reader and thedose-response curve constructed.

Results of In vitro Cytotoxicity Evaluation of the 27 Member PBD LibrarySynthesised on ‘Crowns’

Compound Amino Acid Sequence % Control at 0.3 μM 1 AAA 104 2 AAB 106 3AAC 93.8 4 ABA 86.2 5 ABB 89.6 6 ABC 91.7 7 ACA 87.8 8 ACB 100.6 9 ACC107 10 BAA 112 11 BAB 88.2 12 BAC 99.3 13 BBA 93.9 14 BBB 79.2 15 BBC 9516 BCA 69.6 17 BCB 109 18 BCC 107.6 19 CAA 91.4 20 CAB 99.4 21 CAC 98.322 CBA 85.6 23 CBB 86.1 24 CBC 90.6 25 CCA 119 26 CCB 114 27 CCC 112Benzyl DC-81 — 47.4 A = glycine B = leucine C = phenylalanine

Table 4: In vitro Cytotoxicity of a 27 Member PBD Library Synthesised on‘Crowns’

As before, these results demonstrate that varying the amino acidsequence affects the cytotoxicity of the PBDs.

EXAMPLE 6 DNA Binding Assays

Labelling Double-stranded Oligonucleotides

Double-stranded oligonucleotides (10 pmol/μL) were 5′-end labelled with[³²P]-ATP using T4 polynucleotide kinase and incubated for 30 minutes at37° C. The labelled oligonucleotides were purified through a mini-prepBiorad™ spin column containing P6 Bio-gel™ (40-90 μm).

On-bead Screening Assay

The beads were allowed to swell in DMF for approximately 1 h prior tothe binding experiment. Labelled double-stranded oligonucleotides wereincubated with the beads to which compound was attached for 24 h at 37°C. After 24 h incubation the samples were resuspended in TE buffer (10mM tris, 1 mM EDTA), spun and supernatant removed 3 to 4 times. On thefinal wash the pellet was resuspended in 1 mL of EcoScint (Nat.Diagnostics, UK) scintillation fluid and counted on a Wallac 1400scintillation counter.

Labelled oligonucleotides Oligonucleotide 5′-ACA CCT AIA GAT IAA ITCTI-3′ 1(PuGPu) 3′-TIT IIA TCT CTA CTT CAI AC-5′ Oligonucleotide 5′-ACACCT AIT GTT IAA ITC TI-3′ 2(PyGPy) 3′-TIT IIA TCA CAA CTT CAI AC-5′Oligonucleotide 5′-ACA CCT AIA IAT IAA ITC TI-3′ 3(PuIPu) 3′-TIT IIA TCTCTA CTT CAI AC-5′

Oligonucleotide 1 contains the AGA sequence (highlighted in bold) whichis the most preferred binding site for a PBD. Oligonucleotide 2 containsthe TGT sequence which is the least preferred binding site for a PBD.Oligonucleotide 3 contains the AIA sequence, to which a PBD should notbind owing to the lack of an NH₂ group on the inosine moiety.

Compound JGB-285 is N10-protected and should not bond covalently to DNA:

Compound JGB-285 is a free C10-N11 imine moiety and is able to interactwith DNA:

TABLE 5 Binding of compounds JGB-285 and JGB-286 to Oligonucleotide 1(counts corrected for background)

Compounds Counts per minute (CPM) JGB-285    0 JGB-286 50,394

TABLE 6 Binding of JGB-286 to double stranded oligonucleotides 1, 2, and3 (counts corrected for background) Double-stranded oligonucleotideCounts per minute (CPM) — 59.7 1 50,394 2 3,321 3 1,820

These tests were also carried out using oligonucleotide 1 labelled withRhodamine or Fluorescein instead of [³²P]-ATP (the labelledoilgonucleotides are available from Genesis, Cambridge). Thefluorescence was measured using a Tecan Spectrafluor Plus.

TABLE 7 Binding of compounds JGB-285 and JGB-286 to Rhodamine-labelledOligonucleotide 1 (counts corrected for background). Compounds Relativefluorescent units (RFU) JGB-285  1,122 JGB-286 16,539

TABLE 8 Binding of compounds JGB-285 and JGB-286 to Fluorescein-labelledOligonucleotide 1 (counts corrected for background). Compounds Relativefluorescent units (RFU) JGB-285  1,217 JGB-286 42,355

These results show that the PBD compound on-bead retains its ability tobind covalently to DNA—the protected PBD (JGB-285) did not bind at all,whereas the unprotected PBD (JGB-286) exhibited strong binding. Moreimportantly, the unprotected PBD retained its selectivity for a PuGPusequence, showing little binding activity towards the least preferred,and non-binding sites.

EXAMPLE 7 Synthesis and Screening of Irori™ Glycine PBD Sublibrary (FIG.5) Synthesis

Aminomethylated polystyrene resin 39 (5 g, 1.1 mmol/g loading) wassuspended in DCE: CH₂Cl₂ (2:1, 102 ml) and dispensed equally into 289Irori™ microkans. The resin was filtered and the kans were placed in aflask with DMF (70 ml) and shaken for 30 min.

A solution of Fmoc-glycine (4.91 g, 16.5 mmol), TBTU (5.3 g, 16.5 mmol)and DIPEA (2.9 ml, 16.5 mmol) in DMF (100 ml) was added to the combinedkans and shaking continued for 20 hours. Resin 40 was filtered andrinsed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (3×10 ml) and driedin vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (200 ml) was added to thekans, which were shaken for 16 hours. The acetylated resin was filteredand washed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (3×10 ml) anddried in vacuo.

A solution of 20% piperidine in DMF (200 ml) was added to the acetylatedresin 40 and the reaction flask was shaken for 16 hours. Resin 41 wasfiltered and rinsed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (3×10ml) and dried in vacuo.

The kans were combined and sorted, using the Irori™ software into 17flasks. Each flask contained a solution of an Fmoc-amino acid (0.97mmol), TBTU (312 mg, 0.97 mmol) and DIPEA (170 ml, 0.97 mmol) in DMF (10ml). [Fmoc-alanine (306 mg); Fmoc-asparagine (340 mg,); Fmoc-aspartic(O^(t)Bu) acid (391 mg); Fmoc-glutamine (357 mg); Fmoc-glutamic(O^(t)Bu) acid (408 mg); Fmoc-glycine (289 mg); Fmoc-isoleucine (340mg); Fmoc-leucine (340 mg); Fmoc(Boc)-lysine (357 mg); Fmoc-methionine(459 mg); Fmoc-phenylalanine (374 mg); Fmoc-proline (323 mg);Fmoc-serine (^(t)Bu) (374 mg); Fmoc-threonine (^(t)Bu) (391 mg);Fmoc(Boc)-tryptophan (510 mg); Fmoc-tyrosine (^(t)Bu) (442 mg);Fmoc-valine (323 mg)].

The flasks were shaken for 16 hours and each batch of 17 kans wasfiltered and rinsed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml), MeOH (3×10ml), Et₂O (3×10 ml) and dried in vacuo to give 42.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂(200 ml) was added to all289 kans and the reaction flask was shaken for 16 hours. Acetylatedresin was filtered and washed with CH₂Cl₂(3×10 ml), MeOH (3×10 ml), Et₂O(3×10 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (200 ml) was added to the acetylatedresin and the vessel was shaken for 16 hours. Resin 43 was filtered andrinsed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (3×10 ml) and driedin vacuo.

This process was repeated again to produce a library of trimers 45 andthe final step was carried out simultaneously on all 289 kans.

The kans were placed in a flask with DMF (70 ml) and shaken for 30 min.A solution of Fmoc-PBD acid 7b (Example 1/b) (9.2 g, 16.5 mmol), TBTU(5.3 g, 16.5 mmol) and DIPEA (2.9 ml, 16.5 mmol) in DMF (100 ml) wasadded to the kans and shaking continued for 20 hours. Resin 46 wasfiltered and rinsed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml), MeOH (3×10ml), Et₂O (3×10 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (100 ml) was added to thekans suspended in CH₂Cl₂ (100 ml) and the kans were shaken for 16 hours.The kans were filtered and washed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml),Et₂O (3×10 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (200 ml) was added to the kans andthe reaction flask was shaken for 16 hours. The kans containing resin 47were filtered and rinsed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O(3×10 ml), and dried in vacuo.

Screening

The resulting library was screened against a double stranded DNAsequence to determine which members of the library bound most stronglyto the DNA test sequence.

The DNA sequence used was annealed fluorescein labelled:

Label-5′-ACACCTAIAGATIAAITCTI-3′.

Approximately 10 mg of each library member was placed in a well of a 96well plate and incubated with 5 pmol/μl annealed fluoroscein labelleddouble stranded DNA for 24 hours at 37° C. After 24 hours of incubation,each well was washed 4 times with TE buffer and the beads resuspended in50 mL of TE or PBS.

The fluorescence of each well was measured using a Tecan Spectrafluor todetermine which wells contained the most labelled DNA, and hence whichcompounds bound to the test DNA sequence most strongly.

Most active compounds from the library identified using the Iroriu™software were found to be those with the following combinatorial chains:Gly-Gly-Gln-PBD; Gly-Pro-Iso-PBD; Gly-Thr-Asp-PBD; Gly-Leu-Val-PBD;Gly-Val-Asp-PBD; Gly-Val-Phen-PBD; Gly-Try-Asp-PBD; Gly-Lys-Ala-PBD;Gly-Gly-Asp-PBD; Gly-Gly-Pro-PBD.

EXAMPLE 8 Synthesis of PBD—Glycine Sublibrary (FIGS. 6 a, 6 b, 6 c)Synthesis of Lysine-Glycine Dimer 54 (FIG. 6 a)

Tentagel M NH₂ resin 48 (58 mg, 0.3 mmol/g loading) was weighed into 17Alltech tubes (4 ml volume) and DMF (250 μl) was added to each tube,which were then shaken for 30 min. A solution of Boc(Fmoc) lysine (416mg, 0.88 mmol) in DMF (1.7 μl) and a solution of TBTU (285 mg, 0.88mmol) and DIPEA (155 μl, 0.88 mmol) in DMF (3.4 ml) were equallydispensed into the tubes and shaking continued for 20 hours. Resin 49was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added toeach tube, and the tubes were shaken for 2 hours. The acetylated resinwas filtered and washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), furtherCH₂Cl₂ (3×2 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (250 μl) in CH₂Cl₂ (250 μl)was added to each tube, and the tubes were shaken for 2 hours. Resin 50was filtered and washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), furtherCH₂Cl₂ (3×2 ml) and dried in vacuo.

Resin 50 was suspended in CH₂Cl₂ (250 μl) and shaken for 30 min. An icecold solution of allyl chloroformate (100 μl, 0.88 mmol) and4-methylmorpholine (90 mg, 0.88 mmol) in CH₂Cl₂ (1.7 ml) was equallydispensed to each tube and shaken for 16 hours. Resin 51 was filteredand washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), further CH₂Cl₂ (3×2 ml)and dried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to resin 51 andthe tubes were shaken for 12 hours. Resin 52 was filtered and rinsedwith DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

Resin 52 was suspended in DMF (250 ml) and shaken for 30 min.

A solution of Fmoc-glycine (264 mg, 0.88 mmol) in DMF (1.7 ml) and asolution of TBTU (285 mg, 0.88 mmol) and DIPEA (155 μl, 0.88 mmol) inDMF (3.4 ml) were equally dispensed to the tubes and shaking continuedfor 20 hours. Resin 53 was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂(3×2 ml), MeOH (3×2 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added toresin 53 and the tubes were shaken for 2 hours. The acetylated resin wasfiltered and washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), further CH₂Cl₂(3×2 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to the acetylatedresin and the tubes were shaken for 12 hours. Resin 54 was filtered andrinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried invacuo.

Synthesis of Glycine Sublibrary 61 (FIG. 6 b)

Coupling Conditions

A solution of an Fmoc-amino acid (0.052 mmol) in DMF (100 μl) was addedto each tube containing resin 54. [Fmoc-alanine (16 mg); Fmoc-asparagine(18 mg,); Fmoc-aspartic (O^(t)Bu) acid (21 mg); Fmoc-glutamine (19 mg);Fmoc-glutamic (O^(t)Bu) acid (22 mg); Fmoc-glycine (16 mg);Fmoc-isoleucine (18 mg); Fmoc-leucine (18 mg); Fmoc(Boc)-lysine (24 mg);Fmoc-methionine (19 mg); Fmoc-phenylalanine (20 mg); Fmoc-proline (18mg); Fmoc-serine (^(t)Bu) (20 mg); Fmoc-threonine (^(t)Bu) (21 mg);Fmoc(Boc)-tryptophan (27 mg); Fmoc-tyrosine (^(t)Bu) (24 mg);Fmoc-valine (18 mg)].

A solution of TBTU (285 mg, 0.88 mmol) and DIPEA (155 μl, 0.88 mmol) inDMF (3.4 ml) was equally dispensed into the 17 tubes and shakingcontinued for 20 hours. Resin 55 was filtered and rinsed with DMF (3×2ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacua.

Acetylation Conditions

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added toresin 55 and the tubes were shaken for 2 hours. The acetylated resin wasfiltered and washed with CH₂C₂ (3×2 ml), MeOH (3×2 ml), further CH₂C₂(3×2 ml) and dried in vacuo.

Deprotection Conditions

A solution of 20% piperidine in DMF (500 μl) was added to acetylatedresin and the tubes were shaken for 2 hours. Resin 56 was filtered andrinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried invacuo.

Pool and Split Method

The combined resin 56 was suspended in DCE: CH₂Cl₂ (2:1, 68 ml) in around bottom flask, fitted with a sinter and was aspirated with nitrogengas to ensure thorough mixing. The resin was re-dispensed into the 17Alltech tubes, filtered and DMF (250 μl) was added to each tube,followed by shaking for 30 minutes.

This cycle of coupling, acetylation, deprotection and pooling/splittingwas repeated a further three times, until a library of 6-mer peptides 61had been synthesised.

Synthesis of PBD-Glycine Sublibrary 64 (FIG. 6 c)

The resin 61 was suspended in CH₂Cl₂ (250 μl) and the tubes were shakenfor 30 min. A solution of phenylsilane (876 μl, 7.1 mmol) in CH₂Cl₂ (1.7ml) was equally dispensed into each tube and the tubes were shaken for10 min. A solution of tetrakis(triphenylphosphine)palladium (34 mg, 0.03mmol) in CH₂Cl₂ (1.7 ml) was equally dispensed into each tube and thetubes were shaken for a further 10 min. Resin 65 was filtered and rinsedwith CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), further CH₂Cl₂ (3×2 ml) and driedin vacuo. This procedure was repeated once.

A solution of Fmoc PBD acid 7b (Example 1b) (495 mg, 0.88 mmol) in DMF(3.4 ml) and a solution of TBTU (285 mg, 0.88 mmol) and DIPEA (155 μl,0.88 mmol) in DMF (3.4 ml) were equally dispensed to the suspension of6-mer peptide resin 62 in DMF (250 μl) and the tubes were shaken for 20hours. Resin 63 was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂ (3×2ml), MeOH (3×2 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (250 μl) in CH₂Cl₂ (250 μl)was added to resin 63 and the tubes were shaken for 2 hours. Resin wasfiltered and washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), further CH₂Cl₂(3×2 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to the resin andthe tubes were shaken for 2 hours. Resin 64 was filtered and rinsed withDMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

The resulting sublibrary was screened against rhodamine labelledannealed double strand DNA with the sequence:

Label-5′-ACACCTAIAGATIAAITCTI-3′

The sublibrary was mixed with the DNA sequence (5 pmol/mL) and incubatedat 37° C. for 24 hours with occasional mixing. After 24 hours, thesublibrary was washed 4 times with TE buffer pH 7.6 or PBS. To identifythe beads to which most labelled DNA had bound, agarose gel slides wereprepared as follows. ˜500 mL of 0.25% sea plaque agarose was layeredonto a clean transparent slide and allowed to cool and set. Theincubated beads were then mixed with another ˜500 mL of 0.25% sea plaqueagarose solution and layered onto the precoated slides, and allow tocool and set.

The reddest beads were identifed by eye under a dissecting lightmicroscope, and then retrieved by adding ˜1 mL of water to dried agaroseslide to enable their removal using a p10 gilson pipette with a finetip. The removed beads were then placed into a 1 mL Eppendorf PCR tubeready for identification.

Identification

The identification of the sequences of the most active compounds wascarried out using automated Edman degradation and reversed-phase HPLC.

Pulsed liquid-phase N-terminal sequencing was performed using an AppliedBiosystems (ABI)477A automatic protein sequencer. The selected labelledbeads were loaded onto a glass fibre disc which had previously beenpre-cycled once. The disc was placed in the sequencer and pre-cycledonce, then six cycles of Edman degradation were performed (Edman, P andBegg, G (1967) Eur. J. Biochem. 1, 80). The released phenylthiohydantoin(PTH-) amino acid derivatives were identified by reversed-phase HPLCanalysis. The eight most active compounds were those with the followingsequences:

PBD-KGNNNN; PBD-KGTESF; PBD-KGMPMA; PBD-KGGGMM; PBD-KGKGAS; PBD-KGANIA;PBD-KGMMGG; PBD-KGWYSP

EXAMPLE 9 Synthesis of Split and Mix PED Peptide Library 80 (FIG. 7)

Aminomethylated polystyrene resin VHL 65 (3 g, 1.3 mmol/g loading) wassuspended in DCE: CH₂Cl₂ (2:1, 102 ml) and dispensed equally into 17Alltech tubes (8 ml volume). The resin was filtered and DMF (1.5 ml) wasadded to each tube and shaken for 30 min.

Coupling conditions: (→66)—same as Example 8, but using a solution of anFmoc-amino acid (0.69 mmol) in DMF (250 μl) in each tube. (Fmoc-aminoacids as example 8). The amounts of TBTU and DIPEA were increased inproportion to the amount of Fmoc-amino acid and were in 34 ml of DMF.

Acetylation conditions: same as Example 8, but using 1.5 ml of CH₂Cl₂containing the Ac₂O pyridine.

Deprotection conditions: (→67)same as Example 8, but using 1.5 mlsolution of 20% piperidine in DMF.

Pool and Split method:—same as Example 8, but suspending resin in 102 mlof DCE/CH₂Cl₂.

The cycle was repeated a further four times, until a library of 5-merpeptides had been synthesised.

A solution of Boc(Fmoc) lysine (5.5 g, 11.7 mmol) in DMF (34 ml) and asolution of TBTU (3.76 g, 11.7 mmol) and DIPEA (2.04 ml, 11.7 mmol) inDMF (34 ml) were added to the suspension of 5-mer peptide resin 75 inDMF (17 ml) and the vessel was shaken for 20 hours. Resin 76 wasfiltered and rinsed with DMF (3×5 ml), CH₂Cl₂ (3×5 ml), MeOH (3×5 ml)and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (17 ml) was added to theresin and the vessel was shaken for 2 hours. The acetylated resin wasfiltered and washed with CH₂Cl₂ (3×5 ml), MeOH (3×5 ml), further CH₂Cl₂(3×2 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (17 ml) was added to the acetylatedresin 76 and the vessel was shaken for 12 hours. Resin 77 was filteredand rinsed with DMF (3×5 ml), CH₂Cl₂ (3×5 ml), MeOH (3×5 ml) and driedin vacuo.

A solution of Fmoc PBD acid 7b (Example 1b) (6.55 g, 11.7 mmol) in DMF(34 ml) and a solution of TBTU (3.76 g, 11.7 mmol) and DIPEA (2.04 ml,11.7 mmol) in DMF (34 ml) were added to the suspension of 6-mer peptideresin 77 in DMF (17 ml) and the vessel was shaken for 20 hours. Resin 78was filtered and rinsed with DMF (3×5 ml), CH₂Cl₂ (3×5 ml), MeOH (3×5ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (17 ml) in CH₂Cl₂ (17 ml) wasadded to the resin and the vessel was shaken for 2 hours. Resin 79 wasfiltered and washed with CH₂Cl₂ (3×5 ml), MeOH (3×5 ml), further CH₂Cl₂(3×2 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (17 ml) was added to the resin 79and the vessel was shaken for 2 hours. Resin 80 was filtered and rinsedwith DMF (3×5 ml), CH₂Cl₂ (3×5 ml), MeOH (3×5 ml) and dried in vacuo.

EXAMPLE 10 Synthesis of Glycine Sublibrary 87 (FIG. 8)

Fmoc-aminoethyl photolinker NovaSyn TG resin 20 (30 mg, 0.23 mmol/gloading) was weighed into 17 Alltech tubes (4 ml volume) and a solutionof 20% piperidine in DMF (250 ml) was added to each tube, which wereshaken for 16 hours. Resin 21 was filtered and rinsed with DMF (3×2 ml),CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo. DMF (250 μl) wasadded to each tube and the tubes were shaken for 30 min.

Coupling Conditions:—Same as Example 8, but using solution of anFmoc-amino acid (0.021 mmol) in DMF (150 μl) in each tube. The amountsof TBTU and DIPEA were increased in proportion to the amount ofFmoc-amino acid, and were in 1.7 ml DMF.

Acetylation Conditions:—Same as Example 8

Deprotection Conditions:—Same as Example 8

Pool and Split Method:—Same as Example 8

This cycle of coupling, acetylation, deprotection and pooling/splittingwas repeated twice until a library of trimer peptides 83 had beensynthesised, but after second repetition the resin was not pooled butkept as 17 separate sublibraries, allowing for the final amino acid tobe known.

Coupling to Fmoc-glycine

Resin 83 was suspended in DMF (250 μl) and shaken for 30 min. A solutionof Fmoc-glycine (105 mg, 0.35 mmol) in DMF (1.7 ml) and a solution ofTBTU (112 mg, 0.35 mmol) and DIPEA (68 μl, 0.35 mmol) in DMF (1.7 ml)were dispensed equally to the tubes and shaking continued for 20 hours.Resin was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH(3×2 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added to thetubes and were shaken for 2 hours. The acetylated resin was filtered andwashed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), further CH₂Cl₂ (3×2 ml) anddried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to acetylatedresin and the tubes were shaken for 2 hours. Resin 84 was filtered andrinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried invacuo.

Coupling to Boc(Fmoc)-lysine

A solution of Boc(Fmoc)-lysine (165 mg, 0.35 mmol) in DMF (1.7 ml) and asolution of TBTU (112 mg, 0.35 mmol) and DIPEA (68 μl, 0.35 mmol) in DMF(1.7 ml) were equally dispensed to the tubes and shaking continued for20 hours. Resin was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂ (3×2ml), MeOH (3×2 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added to thetubes and were shaken for 2 hours. The acetylated resin was filtered andwashed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), additional CH₂Cl₂ (3×2 ml)and dried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to acetylatedresin and the tubes were shaken for 2 hours. Resin 85 was filtered andrinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried invacuo.

Coupling to PBD Capping Unit

Resin 85 was suspended in DMF (250 μl) and shaken for 30 min. A solutionof Fmoc-PBD acid 7b (196 mg, 0.35 mmol) in DMF (1.7 ml) and a solutionof TBTU (112 mg, 0.35 mmol) and DIPEA (68 μl, 0.35 mmol) in DMF (1.7 ml)were dispensed equally to the tubes and shaking continued for 20 hours.Resin 86 was filtered and rinsed with DMF (3×2 ml), CH₂Cl₂ (3×2 ml),MeOH (3×2 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (250 μl) and CH₂Cl₂ (250 ml)was added to the tubes which were shaken for 2 hours. Resin was filteredand rinsed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) further CH₂Cl₂ (3×2 ml )and dried in vacuo.

A solution of 20% piperidine in DMF (500 μl) was added to the tubeswhich were shaken for 2 hours. Resin 87 was filtered and rinsed with DMF(3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

EXAMPLE 11 Synthesis of Glycine Sublibrary 103 (FIG. 9)

Aminomethylated resin 88 (30 mg, 0.97 mmol/g loading) was weighed into17 Alltech tubes (4 ml volume). DMF (250 μl) was added to each tube andthe tubes were shaken for 30 min.

Coupling Protocol:—Same as Example 8, but using a solution of anFmoc-amino acid (0.087 mmol) in DMF (250 μl) in each tube. The amountsof TBTU and DIPEA were increased in proportion to the amount ofFmoc-amino acid.

Acetylation Condition:—Same as Example 8

Deprotection Condition:—Same as Example 8

Pool and Split method:—Same as Example 8

This cycle of coupling, acetylation, deprotection and pooling/splittingwas repeated-a further three times until a library of tetramer peptides96 had been synthesised, but after third repetition the resin was notpooled but kept as 17 separate sublibraries, allowing for the finalamino acid to be known.

Coupling to Fmoc-glycine: (96→98)—Same as Example 10 but using 441 mg ofFmoc-glycine in 3.4 ml DMF with the amount of the other compoundsincreased in proportion.

Coupling to Boc(Fmoc)-lysine: (98→100)—Same as Example 10 but using 695mg of TBTU and DIPEA with the amounts of Boc(Fmoc)-lysine in 3.4 ml DMF.

Coupling to PBD Capping Unit: (100→103)—same as Example 10 but using asolution of 828 mg Fmoc PBD acid 7b in 3.4 ml DMF.

EXAMPLE 12 Synthesis of a Bis-PBD Pentapeptide Library Synthesis ofLysine-Glycine Dimer 109 (FIG. 10 a)

Aminomethylated resin 88 (510 mg, 0.97 mmol/g loading) was weighed intoa round bottom flask, fitted with a sinter. DMF (20 ml) was added andthe vessel was shaken for 30 min.

A solution of Boc(Fmoc)-lysine (695 mg, 1.48 mmol) in DMF (10 ml) and asolution of TBTU (480 mg, 1.48 mmol) and DIPEA (260 μl, 1.48 mmol) alsoin DMF (10 ml) were added to the vessel and shaking continued for 20hours. Resin 104 was filtered and rinsed with DMF (3×10 ml), CH₂Cl₂(3×10 ml), MeOH (3×10 ml), Et₂O (2×10 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (20. ml) was added toresin 104 and the vessel was shaken for 2 hours. The acetylated resinwas filtered and washed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O(2×10 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (10 ml) and CH₂Cl₂ (10 ml)was added to the vessel, which was shaken for 2 hours.

Resin 105 was filtered and rinsed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml),Et₂O (2×10 ml) and dried in vacuo.

Resin 105 was suspended in CH₂Cl₂ (5 ml) and shaken for 30 min. An icecold solution of allyl chloroformate (157 μl, 1.48 mmol) and4-methylmorpholine (150 mg, 1.48 mmol) in CH₂Cl₂ (10 ml) was added andthe vessel was shaken for 16 hours. Resin 106 was filtered and rinsedwith CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (2×10 ml) and dried invacuo.

A solution of 20% piperidine in DMF (20 ml) was added to resin 106 andthe tubes were shaken for 2 hours. Resin 107 was filtered and rinsedwith DMF (3×10 ml), CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (2×10 ml) anddried in vacuo.

Resin 107 was suspended in DMF (20 ml) and shaken for 30 min. A solutionof Fmoc-glycine (441 mg, 1.48 mmol) in DMF (10 ml) and a solution ofTBTU (480 mg, 1.48 mmol) and DIPEA (260 μl, 1.48 mmol) in DMF (10 ml)were added to the vessel and shaking continued for 20 hours. Resin 108was filtered and rinsed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml), MeOH (3×10ml), Et₂O (2×10 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (20 ml) was added toresin 108 and the vessel was shaken for 2 hours. The acetylated resinwas filtered and washed with CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O(2×10 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (20 ml) was added to acetylatedresin and the vessel was shaken for 2 hours. Resin 109 was filtered andrinsed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml), MeOH (3×10 ml), Et₂O (2×10ml) and dried in vacuo.

Synthesis of Glycine Sublibrary 117 (FIG. 10 b)

Pool and Split Method:—Same as Example 8, starting with resin 109.

Coupling Conditions:—Same as Example 8 but using a solution of anFmoc-amino acid (0.087 mmol) in DMP (250 μl) in each tube.

Instead of TBTU and DIPEA a solution of diisopropylcarbodiimide (232 μl,1.48 mmol) and HOBt (200 mg, 1.48 mmol) in DMF (3.4 ml) was dispensedequally into the 17 tubes and shaken for 20 hours.

Acetylation Protocol:—Same as Example 8

Deprotection Protocol:—Same as Example 8

This cycle of pooling/splitting, coupling, acetylation and deprotectionwas repeated a further three times until a library of 6-mer peptides 117had been synthesised, but after the third repetition the resin was notpooled but kept as 17 separate sublibraries, allowing for the finalamino acid to be known.

Synthesis of Bis PBD-Glycine Sublibrary 123 (FIG. 10C)

A solution of Boc(Fmoc)-lysine (695 mg, 1.48 mmol) in DMF (3.4 ml) and asolution of TBTU (480 mg, 1.48 mmol) and DIPEA (260 μl, 1.48 mmol) inDMF (3.4 ml) were dispensed equally to resin 117 and shaking wascontinued for 20 hours. Resin 118 was filtered and rinsed with DMF (3×2ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

A solution of 20% Ac₂O, 30% pyridine in CH₂Cl₂ (500 μl) was added toresin 118 and the tubes were shaken for 2 hours. The acetylated resinwas filtered and washed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml), furtherCH₂Cl₂ (3×2 ml) and dried in vacuo.

Resin 118 was suspended in CH₂Cl₂ (250 μl) and the tubes were shaken for30 min. A solution of phenylsilane (1.5 ml, 11.9 mmol) in CH₂Cl₂ (3.4ml) was dispensed equally into each tube and shaken for 10 min.

A solution of tetrakis(triphenylphosphine)palladium (57 mg, 0.05 mmol)in CH₂Cl₂ (3.4 ml) was dispensed equally into each tube and shaken for afurther 10 min. Resin 119 was filtered and rinsed with CH₂Cl₂ (3×2 ml),MeOH (3×2 ml) and dried in vacuo. This procedure was repeated once.

A solution of 20% piperidine in DMF (500 μl) was added to resin 119 andthe tubes were shaken for 2 hours. Resin 120 was filtered and rinsedwith DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

Resin 120 was suspended in DMF (250 μl) and shaken for 30 min. Asolution of Fmoc-PBD acid 7b (1.66 g, 2×1.48 mmol) in DMF (3.4 ml) and asolution of TBTU (960 mg, 2×1.48 mmol) and DIPEA (520 μl, 2×1.48 mmol)in DMF (3.4 ml) were equally dispensed to the tubes and shakingcontinued for 20 hours. Resin 121 was filtered and rinsed with DMF (3×2ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

A solution of 2% triisopropylsilane in TFA (250 μl) and CH₂Cl₂ (250 μl)was added to resin 122 and the tubes were shaken for 2 hours. Resin 122was filtered and rinsed with CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) furtherCH₂Cl₂ (3×2 ml) and dried in vacuo.

A solution of 20% piperidine in DMF (500 ml) was added to resin 122 andthe tubes were shaken for 2 hours. Resin 123 was filtered and rinsedwith DMF (3×2 ml), CH₂Cl₂ (3×2 ml), MeOH (3×2 ml) and dried in vacuo.

EXAMPLE 13 Synthesis of Fmoc-Glu-OAll-Amino-Resin (171) (FIG. 11)

The TentaGel amine resin 169 (3.0 g, 0.8 mmol) in the form of beads wasallowed to swell for 2 hours in dry DMF (12 mL) and shaken gently in asiliconised randomisation round bottom flask, equipped with a sinteredglass filter tube. The suspension was filtered by suction from below.The beads were washed twice with dry DMF as follows: DMF (20 mL) wasadded from the top of the vessel (to wash down any resin adhering to thesides of the vessel), nitrogen gas was gently bubbled from below throughthe sintered glass for 2 minutes, and excess DMF was removed by suction.A threefold molar excess of HOBt (7.7 mL, 0.3 M in DMF, 2.32 mmol)(“coupling reagent” was added to a threefold molar excess ofFmoc-Glu-OAll (0.95 g, 2.32 mmol) and the resulting solution was addedto the resin. A threefold molar excess of PyBOP (1.2 g, 2.32 mmol) and athreefold molar excess of DIPEA (0.4 mL, 0.3 g, 2.32 mmol) in a minimalvolume of DMF (1.2 mL) were added to the reaction flask to initiate thecoupling reaction. The reaction flask was capped tightly and allowed toshake gently for 1 hour at room temperature and excess coupling reagentwas removed by suction. The coupling procedure was repeated once, fromfrom the addition of coupling reagent to ensure complete reaction. Theresulting resin 171 was washed 6 times with DMF (4×20 mL), DCM (1×20 mL)and MeOH (1×20 mL), on each occasion the resin was shaken for 2 minutesand then filtered. The washing cycle was then repeated and the resin wasdried in vacuo.

Synthesis of Pentapeptide Library (172)

In order to cap any remaining free amino groups the resin (in therandomisation flask), was suspended in a mixture of Ac₂O/pyridine/DMF(0.2:0.3:0.5, 10 mL) and allowed to shake for 2 hours. The supernatantwas removed by suction, the beads were washed with DCM (1×10 mL), MeOH(1×10 mL) and again with DCM (1×10 mL). The washing cycle was thenrepeated and the resin was dried in vacuo.

The Fmoc protecting groups were removed by treating the resin with 50%piperidine in DMF (10.7 mL) whilst being shaken. After 10 minutes, thesupernatant was removed by suction and fresh 50% piperidine in DMF (10.7mL) was added and shaking continued. After another 10 minutes, the beadswere washed 6 times with DMF (4×20 mL), DCM (1×20 mL) and MeOH (1×20mL), on each occasion the resin was shaken for 2 minutes and thenfiltered. The washing cycle was then repeated and the resin was dried invacuo.

The beads were suspended in an isopycnic mixture of DCE/DMF (2:1, 32 mL)and nitrogen gas was gently bubbled from below. Equal aliquots (470 μL)of the suspension were added in sequence to each of the 17 Alltechtubes, previously marked with a letter corresponding to a specific aminoacid. This was repeated 4 times. The beads remaining in therandomisation flask were resuspended in the isopycnic mixture (32 mL)and the distribution process was then repeated twice.

The Glu-OAll-resin M1(45.6 mmol) in each Alltech tube was allowed toswell in dry DMF (710 μL) accompanied by gently shaking for 2 hours.Excess DMF was removed by suction on a vacuum manifold.

The resin was washed with dry DMF (1.18 mL) which was added from the topin order to wash down any resin adhering to the side of the Alltechtubes. The tubes were allowed to shake for 2 minutes and excess DMF wasremoved by suction on a vacuum manifold.

A threefold molar excess of HOBt (460 μL, 0.3 M in DMF, 0.137 mmol) wasadded to a threefold molar excess of a each Fmoc-amino-acid (0.137 mmol)and the resulting mixture was shaken for 10 minutes and then added toappropriate Alltech tube.

Amount Substance mg mol TUBE Fmoc-Ala-OH 42.56 0.137 1 Fmoc-Asn-OH 48.50.137 2 Fmoc-Asp(O ^(t)Bu)-OH 56.3 0.137 3 Fmoc-Glu(O ^(t)Bu)-OH 58.20.137 4 Fmoc-Gln-OH 50.4 0.137 5 Fmoc-Gly-OH 40.7 0.137 6 Fmoc-Ile-OH48.3 0.137 7 Fmoc-Leu-OH 48.3 0.137 8 Fmoc-Lys(Boc)-OH 64.1 0.137 9Fmoc-Met-OH 50.8 0.137 10 Fmoc-Phe-OH 51.8 0.137 11 Fmoc-Pro-OH 46.20.137 12 Fmoc-Ser(^(t)Bu)-OH 52.4 0.137 13 Fmoc-Thr(^(t)Bu)-OH 54.40.137 14 Fmoc-Trp(Boc)-OH 72.0 0.137 15 Fmoc-Tyr(^(t)Bu)-OH 62.8 0.13716 Fmoc-Val-OH 46.4 0.137 16

A threefold molar excess of PyBOP (71 mg, 0.137 mmol) and threefoldmolar excess of DIPEA (24 μL, 18 mg, 0.137 mmol) in a minimal volume ofDMF (70 μL), were added to each of the Alltech tubes to initiate thecoupling reaction.

The Alltech tubes were capped tightly and allowed to shake gently for 1hour at room temperature. After this time, excess coupling reagent wasremoved by suction on a vacuum manifold. The coupling procedure wasrepeated once to ensure complete reaction.

The resin was washed 6 times with DMF (4×2 mL), DCM (1×2 mL), MeOH (1×2mL), on each occasion the resin was shaken for 2 minutes and thenfiltered on a vacuum manifold. The washing cycle was then repeated andthe resin was dried in vacuo.

In order to cap any remaining free amino groups in the peptide resin,the resin in each Alltech tube was suspended in a mixture ofAc₂O/pyridine/DMF (0.2:0.3:0.5, 460 μL) and allowed to shake for 2hours. The supernatant was removed by suction on a vacuum manifold andthe beads were washed 3 times with DCM (1×2 mL), MeOH (1×2 mL) and againwith DCM (1×2 mL), on each occasion the resin was shaken for 2 minutesand then filtered on a vacuum manifold. The washing cycle was thenrepeated and the resin was dried in vacuo.

The Fmoc protecting groups were removed by treating the resin with 50%piperidine in DMF (0.62 mL) over 10 minutes on a shaker. The supernatantwas removed and fresh 50% piperidine in DMF (620 μL) was added. Afteranother 10 minutes, the beads were washed 3 times with DMF (4×2 mL), DCM(1×2 mL) and MeOH (1×2 mL), on each occasion the resin was shaken for 2minutes and then filtered on a vacuum manifold. The washing cycle wasthen repeated and the resin was dried in vacuo.

The beads in each of the 17 Alltech tubes were suspended in DCE/DMF(2:1, 1.9 mL), transferred by pipette to the siliconised randomisationflask and excess isopycnic solution removed by suction. The process wasrepeated twice to ensure that all the resin was returned to therandomisation flask.

Once the resin was returned to the randomisation vessel it wasredistributed amongst the 17 Alltech reaction tubes as described above.

The coupling protocol was repeated 4 more times to generate apentapeptide library 172. At the end of the last coupling cycle theresin was not recombined so as to obtain 17 sublibraries, in which theidentity of the N-terminus amino acid was known.

Coupling Library Members to PBD-Capping Unit (172→174)

The peptide resin 172 (45.6 mmol) in each of the Alltech tubes waswashed with DCM (2×2 mL); on each occasion the resin was shaken for 2minutes and then filtered on a vacuum manifold. The resin was then driedin vacuo.

A mixture of CHCl₃/HOAc/NMM (37:2:1, 650 μL) was added to the reactiontubes and shaken for 30 minutes. The deprotected peptide resin waswashed with DCM (4×2 mL), on each occasion the resin was shaken for 2minutes and then filtered on a vacuum manifold. The resin was thenallowed to dry in vacuo.

The peptide resin (45.6 μmol) in each Alltech tubes was allowed to swellin dry DMF (0.8 mL) shaken gently for 2 hours and then filtered usingthe vacuum manifold.

The beads were washed twice with dry DMF as follows: DMF (1.18 mL) wasadded from the top followed by gentle shaking for 2 minutes and excessDMF was removed by filtration on a vacuum manifold.

A threefold molar excess of HOBt (18 mg, 0.137 mmol) in a minimal volumeof DMF (0.3 molar, 700 μL) and threefold molar excess of Alloc-PBD 173synthesised in an analagous manner to example 2 (55 mg, 0.137 mmol) inDMF were added to the resin.

A threefold molar excess of PyBOP (71 mg, 0.137 mmol) and threefoldmolar excess of DIPEA (24 μL, 18 mg, 0.137 mmol) in a minimal volume ofDMF (150 μL), were added to the reaction tube to initiate the couplingreaction.

The reaction tube was capped tightly and allowed to shake gently for 16hours at room temperature. Excess reagents were removed by filtration ona vacuum manifold.

The beads were washed 4 times with DMF (4×2 mL), DCM (1×2 mL) and MeOH(1×2 mL), on each occasion the resin 174 was shaken for 2 minutes andthen filtered on a vacuum manifold. The washing cycle was then repeatedand the resin was dried in vacuo.

Removal of Side Chain, Fmoc and Alloc Protecting Groups (174→175)

The Boc and tBu protecting groups were removed by treating thePBD-peptide resin 174 (45.6 μmol) in each Alltech tubes with a solutionof TFA/triisopropylsilane/DCM (48:2:50, 800 μL). The reaction tubes wereallowed to shake for 30 minutes and excess reagents were removed byfiltration on a vacuum manifold. The procedure was repeated once and thebeads were washed 3 times with DCM (1×2 mL), MeOH (1×2 mL) and againwith DCM (1×2 mL), on each occasion the resin was shaken for 2 minutesand then filtered on a vacuum manifold. The washing cycle was thenrepeated and the resin was dried in vacuo.

The resin was washed with DCM (5×2 mL) on each occasion the resin wasshaken for 30 seconds and then filtered on a vacuum manifold.

Alloc protecting groups were removed by treating the resin (45.6 μmol)with a solution of phenylsilane (PhSiH₃, 130 μL, 0.118 g, 1.09 mmol) inDCM (300 μL) and the resin was stirred manually. A solution of Pd(PPh₃)₄(5.3 mg, 4.56 μmol) in DCM (500 μL) was added, and the Alltech tubeswere shaken mechanically for 10 min. Excess reagents were removed byfiltration on a vacuum manifold and the process was repeated once.

The peptide resin was washed with DCM (8×2 mL), on each occasion theresin was shaken for 30 seconds and then filtered on a vacuum manifold.The washing cycle was repeated and the resin was dried in vacuo.

The Fmoc groups were removed by treating the resin (45.6 μmol) with 50%piperidine/DMF (800 μL) during 2 hours on a shaker. The supernatant wasremoved by filtration on a vacuum manifold.

The beads 175 were washed 6 times with DMF (4×2 mL), DCM (1×2 mL) andMeOH (1×2 mL), on each occasion the resin was shaken for 2 minutes andthen filtered on a vacuum manifold. The washing cycle was repeated twiceand the resin was dried and stored in vacuo.

EXAMPLE 14 Cellulose Paper as a Laminar Support

A. Attachment of an Fmoc-PBD (7b) to Cellulose Paper

Modification of the Cellulose Paper—(General Method)

The required number of points were marked onto a square of cellulosepaper, using a graphite pencil, before the dry paper was incubated withactivated β-alanine solution (12 mL, [0.2 M Fmoc-β-alanine activatedwith 0.24M DIC and 0.4M N-methylimidazole]), for 3 hrs in a sealedvessel. The membrane was washed with DMF (3×50 mL for 3 minutes) andthen treated with piperidine solution for 20 minutes (20% piperidine inDMF, 50 mL).

The membrane was washed with DMF (5×50 mL for 3 minutes) and MeOH (2×50mL, for 3 minutes) and dried.

Fmoc-β-Alanine-OPfp solution (0.3 M Fmoc-βAla-OPfp in DMSO, 1 mL) wascoupled to the pre-defined positions on the membrane. After 15 minutes,the coupling was repeated once again.

The membrane was then acetylated (2 minutes, face-down, in aceticanhydride solution A, 2 mL [2% acetic anhydride in DMF], followed by 30minutes, face-up in acetic anhydride solution B, 50 mL [2% aceticanhydride, 1% DIPEA in DMF], with shaking. After washing with DMF (3×50mL for 3 minutes), the membrane was treated with piperidine solution for20 minutes (20% piperidine in DMF, 50 mL), washed with DMF (5×50 mL for3 minutes) and MeOH (2×50 mL for 3 minutes).

The membrane was stained with bromophenol blue solution (0.01% w/vbromophenol blue in methanol, 50 mL), washed with methanol for 3 minutesand dried.

Coupling of the Fmoc-PBD

A solution of Fmoc-PBD 7b, HOBt and DIC (0.3M, 1.5 mL) in NMP wasspotted at the marked points on the membrane. This was left to couplefor 1 hour and repeated (6×) until the blue colour of the spot wasdischarged. The membrane was washed once with DMF (50 mL) and incubatedwith acetic anhydride solution B, 50 mL for 30 minutes [2% aceticanhydride, 1% DIPEA in DMF].

The membrane was washed with DMF (5×50 mL) for 3 minutes, followed bymethanol (2×50 mL) and dried.

N-Fmoc Deprotection

The Fmoc-protecting group was cleaved by treating the membrane withpiperidine solution (20% in DMF, 20 mL) for 20 minutes.

The membrane was then washed with DMF (5×50 mL) for 3 minutes and MeOH(2×50 mL) for 3 minutes, stained with bromophenol blue solution (50 mL),washed with MeOH (50 mL) for 3 minutes and dried.

[The bromophenol blue colouration was removed by destaining withpiperidine solution, washing with DMF and MeOH and drying.]

B. Attachment of Nvoc-PBD to Cellulose Paper

Coupling of the Nvoc-PBD (7a) (Example 1a)

The general method for modification and attachment of the Fmoc-PBD 7bwas employed. The Nvoc-PBD (7a) was coupled as a 0.3 M solution for 30minutes and repeated (4×) until the bromophenol blue stain wasdischarged. The membrane was washed and dried as previously described.

Deprotection of the Nvoc-protecting Group

The membrane was incubated for several hours in a solution of DMSOcontaining 1% ethanolamine at a wavelength of 365 nm. After washing withDMF (3×50 mL) for 3 minutes and MeOH (2×50 mL) for 3 minutes, themembrane was allowed to dry.

EXAMPLE 15 Synphase Crowns as Solid Support

A. Attachment of Fmoc-PBD to Synphase Crowns

Deprotection of the Fmoc-protecting Group

Two Fmoc-protected Rink-amide crowns (loading 7.7 mM/g), were placed ina scintillation vial and immersed in piperidine solution (20% in DMF, 2mL) for 20 minutes, with shaking. After rinsing with DMF (3×2 mL) andDCM (3×2 mL), the crowns were immersed in a solution of bromophenol blue(0.01% w/v bromophenol blue in MeOH, 5 mL), until equal colouration wasachieved and the crowns were then rinsed in MeOH and dried.

Coupling of the Fmoc-PBD

A solution of Fmoc-PBD 7b (17.18 mg, 3.08×10⁻⁵ mol), HOBt (8.32 mg,6.16×10⁻⁵ mol) and diisopropylcarbodiimide (10.62 mL, 6.16×10⁻⁵ mol) inNMP (500 mL) was added to the scintillation vial with the crowns.

Coupling was monitored by the loss of the bromophenol blue colourationon the crowns. The reaction was allowed to proceed overnight and wasrepeated until complete colour loss had occurred. The crowns were thenwashed with DMF (3×2 mL), DCM (3×2 mL), MeOH (1×2 mL) and allowed toair-dry.

N-Fmoc Deprotection

A single crown was immersed in the piperidine solution (20% in DMF, 1mL) for 20 minutes with shaking. After rinsing with DMF (3×1 mL), andDCM (3×1 mL), the crown was rinsed in MeOH (1×1 mL) and allowed toair-dry.

B. Attachment of Nvoc-PBD (7a) to Synphase Crowns

Coupling of the Nvoc-PBD (7a)

The general method for modification and attachment of the Fmoc-PBD 7bwas employed. The Nvoc-PBD 7a was coupled as a 0.62M solution overnight.

Deprotection of the Nvoc-protecting Group

A single crown was immersed in DMSO containing 1% ethanolamine (2 mL)and irradiated at λ=365 nm for 2 hours, with shaking. After rinsing withDMF (3×1 mL), and DCM (3×1 mL), the crown was rinsed in MeOH (1×1 mL)and allowed to air-dry.

EXAMPLE 16 Synthesis of a 175 Membered Library Utilising Synphase Crownsas the Solid Support (FIG. 12)

Preparation of the Crowns—N-Fmoc Deprotection

175 Rink amide-handle O-series polystyrene crowns 124 were placed intotwo multipin blocks, arranged in a 8×12 format.

The crowns were immersed in the piperidine solution (20% in DMF, 50 mL)for 20 minutes, with shaking. After rinsing with DMF (3×50 mL) and DCM(3×50 mL), the crowns were immersed in a solution of bromophenol blue(0.01% w/v bromophenol blue in methanol, 50 mL), until equal stainingwas achieved and the deprotected crowns 125 were then rinsed in MeOH anddried.

Coupling the first amino acid residue Reagent Mass Solvent Vol. AminoAcid M. Wt. (mg) (mL) Fmoc-Arg(Pbf)-OH 648.8 545.0 8.40 Fmoc-Gly-OH297.3 254.8 8.40 Fmoc-Lys(Boc)-OH 468.5 393.5 8.40 Fmoc-Met-OH 371.5312.1 8.40 Fmoc-Val-OH 339.4 285.1 8.40

Coupling Reagent (used for all couplings in example 16) Reagent M. Wt.Reagent Mass Solvent Vol DIC 126.2 0.530 g 42.0 HOBt 135.1 567.4 mg 42.0

Solutions of the amino acids and coupling reagents detailed above in NMP(200 mL) were dispensed into the deep-well microtiter plates. Couplingswere monitored by the loss of the bromophenol blue staining on thecrowns and were generally allowed to proceed overnight. The crowns 126were then washed in DMF (3×50 mL), DCM (3×50 mL) and allowed to air-dry.

N-Fmoc Deprotection

The crowns 126 were immersed in piperidine solution (20% in DMF, 50 mL)for 20 minutes, with shaking. After rinsing with DMF (3×50 mL) and DCM(3×50 mL) , the deprotected crowns 127 were immersed in a solution ofbromophenol blue (0.01% w/v bromophenol blue in methanol, 50 mL), untilequal staining was achieved and then rinsed in MeOH and dried.

Coupling the second amino acid residue Reagent Mass Solvent Vol. AminoAcid M. Wt. (mg) (mL) Fmoc-Arg(Pbf)-OH 648.8 389.3 6.0 Fmoc-Gly-OH 297.3182.0 6.0 Fmoc-Lys(Boc)-OH 468.5 281.1 6.0 Fmoc-Gln-OH 368.4 221.0 6.0Fmoc-His(Trt)-OH 619.7 379.4 6.0 Fmoc-Leu-OH 353.4 212.0 6.0Fmoc-Tyr(2-ClTrt)-OH 680.2 408.1 6.0

Solutions of the amino acids and coupling reagents detailed above (inNMP, 200 mL) were dispensed into the deep-well microtiter plates.Couplings were monitored by the loss of the bromophenol blue staining onthe crowns 127 and were generally allowed to proceed overnight. Thecoupled crowns 128 were then washed in DMF (3×50 mL), DCM (3×50 mL) andwere allowed to air dry. N-Fmoc deprotection was carried as above, togive crowns 129.

Coupling the third amino acid residue Reagent Mass Solvent Vol. AminoAcid M. Wt. (mg) (mL) Fmoc-Arg(Pbf)-OH 648.8 545.0 8.40 Fmoc-Lys(Boc)-OH468.5 393.5 8.40 Fmoc-Gly-OH 297.3 254.8 8.40 Fmoc-Gln-OH 368.4 309.58.40 Fmoc-Trp-OH 426.5 358.3 8.40

Solutions of the amino acids and coupling reagents detailed above (inNMP, 200 mL) were dispensed into the deep-well microtiter plates.Couplings were monitored by the loss of the bromophenol blue staining onthe crowns and were generally allowed to proceed overnight. The coupledcrowns 130 were then washed in DMF (3×50 mL), DCM (3×50 mL) and allowedto air dry overnight.

N-Fmoc deprotection was carried out as above to give crowns 131.

Coupling the Fmoc-PBD Reagent Mass Solvent Vol. Reagent M. Wt. (g) (mL)Fmoc-PBD 7b 558 2.346 42.0

Fmoc-PBD 7b (2.346 g in 42 ml NHP) and the coupling reagents detailedabove (in NMP, 200 mL) were dispensed into the deep-well microtiterplates. Couplings were monitored by the loss of the bromophenol bluestaining on the crowns 132 and repeated until complete colour loss hadoccurred. The crowns were then washed in DMF (3×50 mL), DCM (3×50 mL)and allowed to air dry.

N-Fmoc Deprotection

The crowns 132 were immersed in piperidine solution (20% in DMF, 50 mL)for 20 minutes, with shaking. After rinsing with DMF (3×50 mL) and DCM(3×50 mL), the crowns 133 were allowed to air dry.

EXAMPLE 17 Use of Rink-amide Resin as Solid Support

A. Attachment of Fmoc-PBD (7b) to Rink-amide Resin

Preparation of the resin—N-Fmoc Deprotection

Fmoc-rink amide resin 200(50 mg, loading 0.63 mmol/g) was

suspended in DCM:DMF (1:1, 1 mL) and shaken for 2 minutes. The resultingsolution was removed under vacuum and the resin resuspended in DMF (1mL) and shaken for 5 minutes.

The DMF was drained from the resin and the Fmoc-group was removed bytreatment with piperidine solution (20% in DMF, 1 mL) for 20 minutes.Excess piperidine solution was removed by suction and the deprotectedresin 201 was washed with DMF and DCM and allowed to dry.

Coupling of the Fmoc-PBD

A solution of Fmoc-PBD propanoic acid 7b (70.3 mg, 1.26×10⁻⁴ mol)diisopropylcarbodiimide (20.1 mL 1.26×10⁻⁴ mol) and HOBt (17.02 mg,1.26×10⁻⁴ mol) in DMF (0.5 mL) was added to the resin 201, and theresulting slurry shaken overnight.

The solution was drained and the coupled resin 202 washed with DMF andDCM. The resin was resuspended in DMF/DCM (DCM) (1 mL) and split intotwo portions. The solutions were removed from both tubes and the resinin the first tube was washed with DCM, methanol and dried under vacuumovernight.

N-Fmoc Deprotection

The resin 202 in the second tube was suspended in piperidine solution(20% in DMF, 0.5 mL) and shaken for 20 minutes. Excess piperidinesolution was drained and the deprotected resin 203 washed with DMF, DCMand methanol, and dried under vacuum overnight.

B. Attachment of Nvoc-PBD (7a)

Preparation of the Resin—N-Fmoc Deprotection

Coupling of the Nvoc-PBD (7a)

The general method for modification and attachment of the Fmoc-PBD 7bwas employed. The Nvoc-PBD 7a was coupled as a 0.25M solution in DMF.

EXAMPLE 18 Attachment of Fmoc-PBD (7b) to Aminoethyl Photolinker AMResin (210)

Preparation of the Resin—N-Fmoc Deprotection

Aminoethyl photolinker AM resin 210(50 mg, 0.21 mmol/g) was placed intotwo Alltech tubes. The resin was suspended in NMP (2 mL) and swelled for2 hours.

After draining excess solvent, the deprotected resin 211 was suspendedin piperidine solution (20% in DMF, 0.5 mL) and shaken for 20 minutes.Excess solvent was drained and the resin washed with DMF, DCM and NMP.

Coupling Protocol of Fmoc-PBD (7b)

A solution of Fmoc-PBD propanoic acid 7b(32.22 mg, 5.78×10⁻⁵

mol, 5.5 equivalents), PyBop (11.4 mg, 2.2×10⁻⁵ mol) and DIPEA (0.05 mL)in NMP (0.5 mL) was added to the resin 211 which was shaken for 1 hour.

After draining, the coupled resin 212 was washed with DCM (5×1 mL),methanol (5×1 mL) and NMP (5×1 mL). The coupling reaction was repeated 3times. The resin in the first tube was then washed with DCM (3×1 mL),methanol (3×1 mL) and water (3×1 mL) and dried in vacuo overnight.

N-Fmoc Deprotection

The Fmoc-PBD resin 212 in the second Alltech tube was re-suspended inpiperidine solution (20% in DMF, 1 mL) and shaken for 20 minutes. Afterdraining, the deprotected resin 213 was washed with NMP (3×1 mL), DCM(3×1 mL), methanol (3×1 mL) and finally water (3×1 mL) and dried invacuo overnight.

EXAMPLE 19 Tentagel-Amino Resin

A. Attachment of Fmoc-PBD (7b) to Novasyn Tentagel Amino Resin (220)

Preparation of the Resin

Novasyn Tentagel amino resin 220 (50 mg, loading 0.28 mmol/g), wasplaced into two Alltech tubes and suspended in DCM:DMF (1:1, 1 mL) andshaken for 2 minutes. Excess solvent was removed by suction and theresin resuspended in DMF (1 mL), shaken for 5 minutes and then drained.

Coupling of FMOC-PBD (7b)

A solution of Fmoc-PBD propanoic acid 7b (23.44 mg, 4.2×10⁻⁵ mol,3×excess), TBTU (13.4 mg, 4.2×10⁻⁵ mol) and DIPEA (7.3 mL, 4.2×10⁻⁵ mol)in DMF (500 mL) was added to the resin 220 and the resulting slurryshaken overnight.

The solution was drained and the resin washed with DMF and DCM. Thecoupling was repeated a second time and the coupled resin 221 washed asbefore. The resin in the first tube was washed further with methanol anddried in vacuo, overnight.

Fmoc Deprotection

The resin 221 in the second tube was suspended in piperidine solution(20% in DMF, 0.5 mL) and shaken for 20 minutes. This solution wasdrained and the deprotected resin 222 washed with DMF, DCM and methanol,and dried under vacuum overnight.

B. Attachment of Nvoc-PBD (7a) to Tentagel Resin

Coupling of the Nvoc-PBD (7a)

The general method for modification and attachment of the Fmoc-PBD 7bwas employed. The Nvoc-PBD 7a was coupled as a 0.085M solution.

Deprotection of the Nvoc-protecting Group

The resin was incubated for several hours in a solution of DMSOcontaining 1% ethanolamine at a wavelength of 365 nm. After washing withDMF (3×50 mL) for 3 minutes and MeOH (2×50 mL) for 3 minutes, themembrane was allowed to dry.

EXAMPLE 20 Synthesis of an PBD-Oligocarbamate Sequence (FIG. 13)

Preparation of the Resin (N-Fmoc Deprotection)

Fmoc-Rink amide resin 134 (53.43 mg, loading 0.63 mmol/g , 3.37×10⁻⁵mol) was suspended in DCM:DMF (1:1, 1 mL) and shaken for 2 minutes.Excess solvent was removed by suction, the resin resuspended in DMF (1mL) and shaken for 5 minutes.

Excess DMF was removed by suction and the Fmoc-group was removed bytreating with piperidine solution (20% in DMF, 1 mL) for 20 minutes. Theresin 135 was then washed with DMF and DCM.

Coupling Protocol Fmoc aminoalkyl No of moles Mass required/ carbonateM. Wt 4× excess mg 1 Fmoc-Lys(Boc)^(c) 619 1.35 × 10⁻⁴ 83.6 2Fmoc-Phe^(c) 539 1.35 × 10⁻⁴ 72.8 3 Fmoc-Ser(^(t)Bu)^(c) 535 1.35 × 10⁻⁴72.2 4 Fmoc-Gly^(c) 448 1.35 × 10⁻⁴ 60.5 5 Fmoc-Tyr(^(t)Bu)^(c) 610 1.35× 10⁻⁴ 82.4 6 Fmoc-Ile^(c) 504 1.35 × 10⁻⁴ 68.0 7 Fmoc-Tyr(^(t)Bu)^(c)610 1.35 × 10⁻⁴ 82.4

A solution of the required 4-nitrophenyl Fmoc-aminoalkyl carbonate, HOBt(36.3 mg) and DIPEA (11 ml), in NMP (200 ml) was added to the free aminoresin 135 and allowed to couple for 4 hours. After draining, the resin136 was washed with NMP and DCM and dried under vacuum.

Deprotection and coupling cycles were repeated until the aminolkylsequence was complete (137-148).

Fmoc-protected PBD (7b) (75.3 mg), HOBt (36.3 mg) anddiisopropylcarbodiimide (21.5 ml) were dissolved in NMP (300 ml) andadded to the free amino resin 148. The slurry was shaken for 24 hours,drained and washed with NMP, DCM and dried overnight, under vacuum togive PBD resin 149. This was deprotected as above to give unprotectedPBD resin 150.

Cleavage from the Resin

The resins (149 and 150) were treated with a solution of 95% TFA:5%water:2.5% triisopropylsilane (1 mL) at RT for 2 hours. The resins wereremoved by filtration, washed with a small amount of TFA and DCM. Thesolvent was removed in vacuo and the residue dissolved inacetonitrile:water 1:2 and lyophilised twice.

EXAMPLE 21 Synthesis of a 289 Member Library Using 4-nitrophenyl N-FmocAminoalkyl Carbonates (FIG. 14)

Formation of N-(9-fluorenylmethoxycarbonyl) amino alcohols. (Generalmethod of Cho et al: Synthesis and Screening of Linear and CyclicOligocarbamate Libraries. Discovery of High Affinity Ligands forGPIIb/IIIa, J. Am. Chem. Soc., (1998), 120, 7706-7718., C. Y. Cho, R. S.Youngquist, S. J. Paikoff, M. H. Beresini, A. R. Herbert. L. T. Berleau,C. W. Liu, D. E. Wemmer, T. Keouah and P. G. Schultz.)

The appropriate N-Fmoc-protected amino acid (10 mmol) anddimethoxyethane were stirred under nitrogen, in an ice/salt bath.N-methylmorpholine (1.11 mL, 10 mmol) and isobutylchloroformate (1.36mL, 10 mmol) was added to the solution.

After stirring for 1 min, under nitrogen, the solid was removed byfiltration and sodium borohydride (570 mg, 15 mmol) in water (20 mL),was added to the filtrate. Additional water (150 mL) was added after 20minutes, and the solution allowed to stir for 1 hour at room temp.

The precipitated product was filtered and washed with a small amount ofwater followed by hexane. The solid was redissolved in ethyl acetate,dried using MgSO₄ and the solvent removed in vacuo.

For those amino alcohol derivatives that did not precipitate fromsolution, the solution was extracted with ethyl acetate (5×300 mL). Theorganic extract was dried, over MgSO₄ and the solvent removed in vacuo.

Fmoc-amino alcohols were used without further purification—the data forthe amino alcohols are shown in Appendix 1

Formation of 4-nitrophenyl N-(9-fluorenylmethoxycarbonyl)-aminoalkylCarbonates (General Method of Cho et al.)

To the appropriate N-Fmoc-amino alcohol (10 mmol) was added pyridine (11mmol) and DCM (50 mL). A solution of 4-nitrophenyl chloroformate (11mmol) in DCM (10 mL) was added dropwise to the reaction mixture. Themixture was stirred for at least 24 hours. The mixture was diluted withDCM (100 mL) and washed with 1.0 M sodium bisulfate (3×75 mL) and 1.0 Msodium bicarbonate (10×100 mL). The organic layer was dried, using MgSO₄and the solvent removed in vacuo.

The crude product was purified using silica gel chromatography (9:1DCM:hexane DCM). The data for the amino alkyl nitrophenyl carbonate isshown in Appendix 2.

Library Synthesis

Preparation of the Resin

Rink amide MBHA resin 151 (8.67 g, loading 0.54 mMol/g) was suspended ina solution of DCM/DMF (3:1) and distributed between 289 Irori MicroKanreactors containing RF tags.

The Kans were immersed in a solution of DCM/DMF (1:1, 300 mL) and shakenvigorously for 1 min. The solvent was removed under vacuum and the Kanswere re-immersed in DMF and shaken vigorously for 10 minutes.

The DMF was drained and the Fmoc-group was removed by adding piperidinesolution (20% in DMF, 300 mL) to the Kans followed by shaking for 5minutes and draining. Additional piperidine solution was added (20% inDMF) and the Kans were shaken for 1 hour. The Kans with deprotectedresin 152 were drained and washed with DMF (6×300 mL) and DCM (3×300mL).

Coupling of the First 4-nitrophenyl Fluorenylmethoxy AminoalkylCarbonate

A solution of Fmoc-Valine^(c) (6.88 g, 4.86×10⁻⁵ mol, 3×excess), HOBt(3.795 g, 9.72×10⁻⁵ mol) and DIPEA (1.22 mL, 2.43×10⁻⁵ mol) in NMP (300mL) was added to the Kans and allowed to couple for 4 hours.

After draining the Kans with coupled resin 153 were washed with NMP(3×300 mL) and DCM (3×300 mL).

Fmoc-deprotection

The Fmoc-protecting group was removed by adding piperidine solution tothe Kans (20% in DMF; 300 mL) followed by shaking for 1 hour. The Kanswith deprotected resin 154 were drained and washed with DMF (3×300 mL)and DCM (3×300 mL).

Coupling of the second 4-nitrophenyl fluorenylmethoxy aminoalkylcarbonate Fmoc-Amino alkyl nitrophenyl carbonate Mass (mg) Ala 382 Asn417 Asp(O^(t)Bu) 467 Arg(Pbf) 673 Gln 428 Glu(O^(t)Bu) 449 Gly 370 Ile416 Leu 416 Lys(Boc) 511 Met 430 Phe 445 Pro 477 Ser(^(t)Bu) 442Thr(^(t)Bu) 453 Trp 477 Tyr(^(t)Bu) 504

The Kans with the resin 154 were sorted using their Rf tags into 17flasks containing solutions of the appropriate 4-nitrophenyl Fmoc-aminoalkyl carbonate (8.262×10⁻⁴ mol), HOBt (223 mg, 1.6524×10⁻³ mol) andDIPEA (71.4 mL, 4.131×10⁻⁴ mol) in NMP (15 mL). The Kans were agitatedfour 4 hours, drained and washed with NMP (3×100 mL) and DCM (3×100 mL),and then contained coupled resin 155).

N-Fmoc Deprotection

The 289 Kans with coupled resin 155 were pooled in one flask and theFmoc-protecting group was removed by treatment with piperidine solution(20% in DMF, 300 mL) for 1 hour. The Kans with deprotected resin 156were drained and washed with DMF (3×300 mL) and DCM (3×300 mL).

Coupling of the third 4-nitrophenyl fluorenylmethoxy aminoalkylcarbonate Fmoc-Amino alkyl nitrophenyl carbonate Mass (mg) Ala 382 Asn417 Asp(O^(t)Bu) 467 Arg(Pbf) 673 Gln 428 Glu(O^(t)Bu) 449 Gly 370 Ile416 Leu 416 Lys(Boc) 511 Met 430 Phe 445 Pro 477 Ser(^(t)Bu) 442Thr(^(t)Bu) 453 Trp 477 Tyr(^(t)Bu) 504

The Kans with resin 156 were sorted via their Rf tags into 17 flaskscontaining solutions of the appropriate 4-nitrophenyl Fmoc-amino alkylcarbonate (8.262×10⁻⁴ mol) , HOBt (223 mg, 1.6524×10⁻³ mol) and DIPEA(71.4 ml, 4.131×10⁻⁴ mol) in NMP (15 mL). The resin in the Kans wasallowed to couple for 4 hours to from coupled resin 157. After draining,the Kans were washed with NMP (3'100 mL) and DCM (3×100 mL).

N-Fmoc Deprotection was carried out as above to give resin 158 in theKans.

Coupling of the Fmoc-PBD (7b)

A solution of Fmoc-PBD (7b) (7.84 g, 1.40×10⁻² mol, 3×excess), HOBt(1.896 g, 1.40×10⁻² mol) and diisopropylcarbodiimide (2.18 mL, 1.40×10⁻²mol) in NMP (300 mL) was added to the 289 Kans with resin 158. The Kanswere stirred vigorously for 24 hours and drained. The Kans which thencontained coupled resin 159 were washed with NMP (3×300 mL) and DCM(3×300 mL).

N-Fmoc deprotection was carried out as before to give resin 160 in theKans.

EXAMPLE 22 Synthesis of a 27 Member Library Using Peptoids (IroriMethod) (FIG. 15)

Preparation of the Resin

Rink amide MBHA resin 151 (810 mg, loading 0.54 mMol/g) was suspended ina solution of DCM/DMF (3:1, 5.4 mL) and distributed between 27 IroriMicroKan reactors containing RF tags.

The Kans were immersed in a solution of DCM/DMF (1:1, 50 mL) and shakenvigorously for 1 min. This solution was removed under vacuum and theKans were re-immersed in DMF and shaken vigorously for 10 minutes.

The DMF was drained and the Fmoc-group of resin 151 was removed byadding piperidine solution (20% in DMF, 50 mL) to the Kans. This wasshaken for 5 minutes and drained. Further piperidine solution was added(20% in DMF, 50 mL) and the Kans shaken for 1 hour. The Kans withdeprotected resin 152 were drained and washed with DMF (6×50 mL).

Acylation Step

Bromoacetic acid solution (0.6M in DMF, 60 mL) was added to the Kanswith resin 152, followed by diisopropylcarbodiimide solution (3.2M inDMF, 14.1 mL). This was shaken for 2 hours at room temperature, drainedand repeated. The Kans with bromoacetamide resin 161 were then washedwith DMF (2×50 mL) and DMSO (1×50 mL).

Displacement Step

The 27 Kans were sorted via their RF tags into 3 flasks. The first setwere suspended in aq. methylamine solution (40% w/v, 20 mL), the secondset in piperonylamine solution (2M in DMSO, 20 mL) and the third set in2-methoxyethylamine (2M in DMSO, 20 mL). These were shaken vigorouslyfor 4 hours and then drained. The Kans with amino-coupled resin 162 werewashed with DMSO (2×20 mL),and DMF (1×20 ml).

The acylation and displacement steps were repeated twice to give resin166 (which is a library of 27 resins with all combinations of 3 valuesfor R₁, R₂ and R₃)

Coupling of the Fmoc-PBD (7b)

The 27 Kans with the peptoid bearing resin 166 were re-combined and asolution of the Fmoc-PBD (7b) (976.3 mg, 1.75 mmol, 4×excess), HOBt(236.4 mg, 1.75 mmol) and diisopropylcarbodiimide (22.08 mg, 1.75 mmol)in DMF (20 mL) added. This was stirred vigorously for 24 hours anddrained. The Kans with the Fmoc-PBD coupled resin 167 were washed withDMF (3×50 mL) and DCM (3×50 mL).

N-Fmoc Deprotection

The Fmoc-protecting group was removed from the PBD resin 167 by addingpiperidine solution (20% in DMF, 50 mL) to the Kans. This was shaken for5 minutes and drained. Further piperidine solution was added (20% inDMF, 50 mL) and the Kans shaken for 1 hour. The Kans with thedeprotected resin 168 were drained and washed with DMF (3×50 mL), DCM(3×50 mL), methanol (2×50 mL) and dried overnight under vacuum.

APPENDIX 1

Data for Amino Alcohols Incl Yield (%) &NMR

Ala:−74%

¹H NMR (270 MHz, d₆-acetone): δ7.88 (d, 2, J=7.1 Hz), 7.71 (d, 2, J=7.3Hz), 7.31-7.42 (dt, 4), 7.09 (d, 1, J=8.1 Hz), 4.28 (m, 4), 3.54 (m, 1),3.36 (m, 1), 3.26 (m, 1), 1.04 (d, 3, J=6.6 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ155.5, 143.9 140.7 127.5, 127.0, 0.1,120.0, 65.1 64.4, 48.4 46.7 17.3

Arg(Pbf):

¹H NMR (270 MHz, d₆-acetone): δ, 7.81 (d, 2, J=7.7 Hz), 7.66 (d, 2,J=7.3 Hz), 7.29-7.38 (dt, 4), 6.54 (s, br, 1), 4.31 (m, 2), 4.18 (t, 1,J=6.95 Hz), 4.06 (q, 2, J=6.95 Hz), 3.63 (m, 1), 3.53 (m, 2), 3.21 (m,2), 2.94 (m, 2), 2.60 (s, 4), 2.51 (s, 3), 1.96 (s, 2), 1.38 (m, 10),1.19 (t, 2, J=7 Hz), 0.89 (m,1).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 158.9, 157.3, 145.1 145.0, 138.7,135.3, 132.8, 128.4, 127.9, 126.1, 125.3, 120.7, 117.5, 86.9, 66.8,62.3, 60.5, 53.7, 48.1, 43.6, 41.6, 20.8, 19.5, 18.2, 14.5, 12.5.

Asp(OtBu):−84%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.5 Hz), 7.69 (d, 2,J=7.3 Hz), 7.30-7.45 (dt, 4), 7.16 (d, 1, J=8.8 Hz), 4.84 (m, 1), 4.31(m, 3), 3.87 (m,1), 3.39 (m, 2), 2.26 (m,1), 1.37 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 170.4, 155.5, 143.8, 140.7, 127.5,127.0, 125.1,120.0, 79.6,65.2, 62.9, 50.2, 46.7, 27.6.

Asn:−56%

¹H NMR (270 MHz, d₆-acetone): δ, 7.96, 7.88 (s, d, 2, J=7.5 Hz), 7.70,(m, 2), 7.31-7.43 (dt, 4), 4.34 (m, 4), 3.74 (m, 3), 3.62 (t, 1, J=4.6Hz), 3.44 (m, 1), 2.52 (m, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 162.3, 155.6, 143.7, 140.6, 127.6,125.2, 125.0, 120.0, 65.2, 54.1, 46.6, 46.5.

Gln:−86%

¹H NMR (270 MHz, d₆-acetone): δ, 7.96, 7.87 (s, d, 3, J=7.3 Hz), 7.70(d, 2, J=7.1 Hz), 7.30-7.42 (dt, 4), 7.13 (d, 1, J=8.6 Hz), 4.74 (s, br,1), 4.24 (m, 3), 3.43 (m, 5), 2.89 (s, 2), 2.73 (s, 2), 2.45 (m, 3),2.03 (s, 3), 1.80 (m, 1), 1.59 (m ,1).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 162.2, 155.9, 143.9, 143.8, 140.7,127.5, 126.9, 125.1, 124.7, 120.0, 65.1, 63.2, 52.0, 46.7, 35.7, 30.5,29.9, 14.6.

Glu(OtBu):−71%

¹H NMR (270 MHz, d₆-acetone) δ, 7.88 (d, 2, J=7.3 Hz), 7.71 (d, 2, J=7.3Hz), 7.31-7.45 (dt, 4), 7.05 (d, 1, J=8.6 Hz), 4.24 (m, 3), 4.02 (q, 1,J=7.1 Hz), 3.37 (m ,2), 2.20 (m, 2), 1.40 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 172.1, 155.9, 143.9, 143.8, 140.7,127.5, 126.7, 125.2, 120.0, 79.3, 65.1, 63.3, 52.1, 46.8, 31.5, 27.7,26.3

Gly:−79%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.1 Hz), 7.69 (d, 2,J=7.1 Hz), 7.24-7.42 (m, 5), 4.66 (t, 1, J=5.5 Hz), 4.29 (m, 3), 3.38(m, 2), 3.09 (q, 2, J=6 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 156.2, 143.9, 140.7, 127.5, 127.0,125.1, 120.0, 65.2, 59.8, 46.7, 43.0.

Ile:−72%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.1 Hz), 7.73 (d, 2,J=7.3 Hz), 7.29-7.42 (m, 4), 7.03 (d, 1, J=8.4 Hz), 4.51 (m, 1), 4.23(m, 3), 3.42 (m, 3), 0.84 (m, 6).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 156.1, 144.0, 143.8, 140.7, 127.5,126.9, 125.2, 120.0, 65.1, 61.1, 57.0, 46.8, 35.3, 24.6, 15.4, 11.3.

Leu:−88%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.5 Hz), 7.71 (d, 2,J=7.3 Hz), 7.30-7.44 (dt, 4), 6.99 (d, 1, J=8.8 Hz), 4.24 (m, 3), 3.54(m, 1), 3.35 (m, 2), 1.62 (m, 1), 1.30 (m, 2), 0.87 (m, 6).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.9, 144.0, 143.8, 140.7, 127.5,126.9, 125.2, 125.2, 120.0, 65.0, 64.1, 50.9, 46.8, 24.2, 23.4, 21.7.

Lys(Boc):−95%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.7 Hz), 7.72 (d, 2,J=7.0 Hz), 7.30-7.42 (dt, 4), 4.23 (m,3), 4.01 (q, 1, J=7.0 Hz), 2.89(m, 2), 1.37 (m, 12), 1.17 (t, 2, J=7.1 Hz), 0.89 (d, 1, J=6.6 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.9, 155.5, 143.9, 143.9, 143.3,140.7, 127.7, 127.5, 127.0, 125.1, 120.0, 77.2, 65.1, 63.5, 59.7, 52.8,46.7, 46.6, 29.5, 28.2, 22.8, 20.7, 18.7, 14.0.

Met:−52%

¹H NMR (270 MHz, d₆-acetone): δ, 7.91, 7.72 (m, d, 4, J=6.6 Hz), 7.34(m, 4), 7.08 (d, 1, J=8,1 Hz), 6.75 (m, 1), 4.28 (m, 3), 3.42 (m, 3),2.89 (s, 2), 2.74 (s, 2), 2.11 (m, 2), 1.81 (m, 1), 1.56 (m, 1).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 162.2, 155.9, 143.9, 140.7, 127.6,127.0, 125.2, 120.0, 119.9, 65.2, 63.3, 52.6, 46.7, 26.8.

Phe:−94%

¹H NMR (270 MHz, d₆-acetone): δ, 7.87 (d, 2, J=7.3 Hz), 7.65 (m, 2),7.24-7.41 (m, 10), 4.81 (t, 1, J=5.5 Hz), 4.16 (m, 3), 3.67 (m, 1), 3.39(m, 2), 2.87 (dd, 1, J=4.9, 8.6 Hz), 2.67 (m, 1).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.6, 143.8, 140.6, 139.2, 1291,128.0, 127.5, 127.0, 125.8, 125.2, 125.1, 120.0, 65.1, 62.9, 54.6, 46.6.

Pro:−73%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=6.7 Hz), 7.65 (d, 2,J=7.3 Hz), 7.31-7.43 (dt, 4), 4.74 (m, 1), 4.30 (m, 3,), 3.73 (m, 1),3.27 (m, 3,), 1.86 (m, 4), 1.17 (t, 1, J=7.1).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 154.1, 143.9, 140.8, 127.6, 127.0,125.0, 120.0, 66.4, 66.2, 61.8, 61.1, 59.7, 58.9, 58.3, 46.8, 46.3,27.7, 26.9, 23.2, 22.4, 20.7, 14.0.

Ser(tBu):−76%

¹H NMR (270 MHz, d₆-acetone): δ, 7.88 (d, 2, J=7.5 Hz), 7.11 (d, 2,J=7.3 Hz), 7.30-7.45 (dt, 4), 6.99 (d, 1, J=8.1 Hz), 4.24, 3.74,3.30-3.56 (m, m, m, 11), 1.12 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.9, 144.0, 143.9, 140.8, 127.6,127.1, 125.3, 125.3, 120.1, 72.4, 65.3, 60.7, 53.6, 46.8, 27.4, 18.9.

Thr(tBu ):

¹H NMR (270 MHz, d₆-acetone): δ, 7.90 (d, 2, J=7.3 Hz), 7.70 (d, 2,J=7.3 Hz), 7.28-7.44 (dt, 4), 6.89 (d, 1, J=8.1 Hz), 4.23 (m, 3), 3.68(m, 1), 3.29 (m, 1), 2.73 (m, 1), 1,15 (m, 12).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.6, 143.9, 143.7, 140.8, 127.5,127.1, 125.2, 125.2, 120.2, 72.3, 65.3, 60.7, 53.6, 46.8, 27.3, 18.6,17.9.

Trp:−89%

¹H NMR (270 MHz, d₆-acetone): δ, 10.81 (s, 1), 7.86 (d, 2, J=7.3 Hz),7.67 (m, 3), 7.34 (m, 5), 6.96-7.14 (m, 4), 4.76 (t, 1, J=5.3 Hz), 4.24(m, 3), 3.77 (m, 1), 3.41 (m, 2), 2.94, 2.5 (m, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.8, 143.9, 140.6, 136.1, 127.5,127.5, 127.0, 125.2, 125.2, 123.1, 120.7, 112.0, 118.4, 118.1, 111.4,111.2, 65.2, 62.8, 53.8, 46.7, 26.7.

Tyr(tBu):−88%

¹H NMR (270 MHz, d₆-acetone): δ, 7.87 (d, 2, J=7.3 Hz), 7.66 (d, 2,J=7.3 Hz), 7.31-7.41 (m, 4), 7.11 (m, 3), 6.82 (d, 2, J=8.2 Hz), 4.79,(t, 1, J=5.5 Hz), 4.13 (m, 3), 3.64 (m, 1), 3.38 (m, 1), 2.82 (dd, 1,J=4.8, 9.0 Hz), 1.19 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 155.6, 153.0, 143.9, 143.8, 140.6,133.8, 129.5, 127.5, 126.9, 125.2, 125.1, 123.3, 112.0, 77.4, 65.2,63.0, 54.6, 46.6, 28.4.

Val:−96%

¹H NMR (270 MHz, d₆-acetone) δ, 7.88 (d, 2, J=7.3 Hz), 7.73 (d, 2, J=7.3Hz), 7.32-7.41 (dt, 4), 4.24 (m, 3), 3.89 9d, 1, J=6.2 Hz), 3.39 (m, 2),0.86 (m, 6), 0.54 (t, 1, J=7.1 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 156.3, 152.7, 143.9, 143.9, 140.7,127.5, 126.9, 125.2, 120.0, 65.2, 61.4, 57.9, 46.8, 46.2, 28.4, 19.5,18.6, 17.9.

APPENDIX 2

Data forAmino Alkyl Nitrophenyl Carbonates Incl Yield (%), NMR (d/ppm,d₆-acetone).

Ala:,

¹H NMR (270 MHz, d₆-acetone): δ, 8.32 (d, 2, J=9.1 Hz), 7.85 (d, 2,J=7.3 Hz), 7.71 (d, 2, J=7.3 Hz), 7.56 (d, 2, J=9.3 Hz), 7.30-7.41 (m,7), 4.07-4.44 (m, 6), 1.24 (d,3, J=6.6 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.4, 156.7, 156.5, 153.1, 146.1,145.0, 144.9, 141.9, 128.3, 127.8, 126.0, 125.9, 123.1, 120.7, 72.1,66.5, 47.7, 46.3, 17.1.

Arg(Pbf):

¹H NMR (270 MHz, d₆-acetone): δ, 8.13 (d, 2, J=4.9 Hz), 7.68 (d, 4, 7.7Hz), 7.52 (d, 4, J=7.8 Hz), 7.24-7.37 (d, 10, J=9.3 Hz), 4.07-4.22 (m,6), 3.87 (m, 9, 1), 3.37 (m, 2), 2.47 (d, 6, J=3.1 Hz), 1.26 (m, 12).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 207.3, 160, 158.3, 157.7, 154.3,147.4, 146.2, 146.1, 145.9, 143.1, 139.8, 136.5, 133.9, 129.5, 129.5,128.9, 127.0, 124.1, 121.8, 121.8, 88.0, 80.2, 72.5, 67.9, 67.8, 66.1,49.2, 49.1, 44.6, 29.7, 27.8, 20.5, 19.3, 19.2, 13.6.

Asp(OtBu):−74%

¹H NMR (270 MHz, d₆-acetone): δ, 8.31 (d, 2, J=9.5 Hz), 7.86 (d, 2,J=7.7 Hz), 7.54 (d, 2, J=9.1 Hz), 7.39 (m, 6), 4.37 (m, 4), 4.24 (m, 1),4.06 (q, 1, J=6.9 Hz), 2.66 (m, 1), 1.46 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.1, 170.3, 156.7, 153.2, 146.4,145.0, 142.1, 128.5, 126.8, 126.0, 123.1, 120.8, 81.3, 70.7, 67.1, 48.2,37.9, 28.2.

Asn:10%

¹H NMR (270 MHz, d₆-acetone): δ, 8.31 (d, 1, J=9.5 Hz), 8.14 (m, 2),7.98 (m, 1), 7.83 (d, 1, J=7.3 Hz), 7.67 (t, 1, J=8.1 Hz), 7.53 (d, 1,J=9.2 Hz), 73.0-7.39 (dt, 3), 6.99 (m, 2), 4.41 (m, 2), 4.24 (m, 1),2.94 (m, 2), 2.79 (s, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.3, 164.4, 163.1, 156.8, 156.6,153.1, 144.9, 142.1, 141.6, 128.5, 127.9, 127.8, 126.1, 123.1, 120.8,116.5, 79.2, 73.8, 69.6, 67.3, 48.0, 36.3, 20.6.

Gln:−17%

¹H NMR (270 MHz, d₆-acetone): δ, 8.31 (dd, 2, J=2.7 Hz, 5.5 Hz), 8.169dd, 1, J=2.2 Hz, 4.8 Hz), 7.83 (d, 2, J=7.3 Hz), 7.68 (m, 2), 7.27-7.54(dt, m, 6), 7.03 (dd, 1, J=2.2 Hz, 4.8 Hz), 6.65 (d, 1, J=8.4 Hz),4.21-4.46 (m, 5), 2.59 (m, 2), 1.92 (m, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.2, 157.1, 156.7, 153.3, 145.1,144.9, 142.1, 128.5, 127.9, 126.8, 126.3, 123.1, 120.8, 116.6, 71.3,66.9, 50.2, 48.1, 15.3.

Glu(OtBu):−76%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=9.1 Hz), 7.83 (d, 2,J=7.3 Hz), 7.69 (m, 2), 7.50 (d, 1, J=6.9 Hz), 7.39 (m, 4), 4.39 (m, 2),4.26 (m, 2), 4.06 (m, 1), 2.38 (m ,1), 1.44 (s, 9).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.2, 172.6, 157.1, 156.7, 153.2,146.3, 145.1, 144.9, 142.1, 128.5, 127.9, 126.0, 123.1, 120.8, 80.5,71.4, 66.9, 50.4, 49.0, 32.3, 27.0.

Gly:−30%

¹H NMR (270 MHz, d₆-acetone): δ, 8.29 (d, 2, J=9.1 Hz), 7.84 (d, 2,J=7.3 Hz), 7.68, (d, 2, J=7.3 Hz), 7.54, (d, 2, J=9.1 Hz), 7.31-7.4,(dt, 4), 4.39 (m, 4), 4.24, (t, 1, J=6.9 Hz), 3.56, (q, 2, J=5.1 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.1, 157.4, 156.7, 153.3, 146.4,145.1, 142.1, 128.5, 127.9, 126.3, 126.0, 123.1, 120.8, 68.8, 67.1,48.1, 40.4.

Ile:−91%

¹H NMR (270 MHz, d₆-acetone): δ, 8.26 (d, 2, J=9.2 Hz), 7.82 (d, 2,J=7.5 Hz), 7.68 (m, 2), 7.50 (d, 2, J=9.1 Hz), 7.26-7.47 (dt, 3),4.20-4.51 (m, 3), 3.92 (m, 1), 1.60 (m, 1), 1.19 (m, 1), 1.01, 0.92 (d,t, 3, J=6.8 Hz, 7.3 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.3, 156.7, 153.3, 145.4, 144.1,142.1, 128.5, 127.9, 127.8, 126.1, 126.0, 123.1, 120.8, 70.2, 66.9,54.9, 54.8, 48.1, 36.7, 15.7, 11.4.

Leu:−88%

¹H NMR (270 MHz, d₆-acetone): δ, 8.27(d, 2, J=9.1 Hz), 7.83 (d, 2, J=7.3Hz), 7.67 (t, 2, J=5.8 Hz), 7.51 (m, 2,), 7.29-7.49 (dt, 4), 4.38 (m,3), 4.22 (m, 3), 1.77 (m, 1), 1.55 (m, 1), 1.37 (m, 1), 0.94 (m, 6).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 205.4, 156.4, 155.9, 152.5, 145.6,144.4, 144.2, 141.3, 127.7, 127.1, 127.1, 125.2, 122.3, 112.0, 71.3,66.1, 48.2, 47.3, 24.5, 22.7, 21.3.

Lys(Boc):−64%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (m ,1), 7.86 (m, 2), 7.52 (m, 2),7.31-7.40 (m, 5), 4.23-4.37 (m, 3), 3.08 (m, 2), 1.39 (m, 14).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.2, 157.2, 156.7, 146.4, 145.1,145.0, 142.1, 128.5, 127.9, 126.8, 126.0, 123.7, 123.1, 120.8, 116.5,78.4, 71.6, 67.3, 66.9, 50.9, 48.1, 48.0, 40.8, 40.7, 23.8.

Met:−13%

¹H NMR (270 MHz, d₆-acetone): δ, 8.29 (d, 2, J=9.1 Hz), 7.83 (d, 2,J=7.3 Hz), 7.69 (d, 2, J=7.3 Hz), 7.39 (m, 4), 4.17-4.42 (m, 3),3.60-3.71 (m, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 205.5, 156.4, 144.4, 144.2, 141.3,127.7, 127.7, 125.3,125.3, 122.3, 120.0, 70.6, 66.0, 50.8, 47.4, 29.3,29.1, 13.5.

Phe:−36.%

¹H NMR (270 MHz, d₆-acetone): δ, 8.28 (d, 2, J=9.1 Hz), 7.82 (d, 2,J=7.5 Hz), 7.62 (m, 2), 7.51, 7.38-7.18 (d, m, m, 10, J=9.1 Hz), 6.75(d, 1, J=7.9 Hz), 4.46 (d, 1, J=6.59), 4.31 (m, 3), 4.18 (m, 1), 2.97(m, 2).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.2, 156.9, 156.7, 153.2, 146.4,145.0, 142.0, 138.8, 130.1, 129.2, 128.4, 127.9, 127.3, 126.8, 126.0,123.1, 120.7, 70.9, 66.9, 52.4, 48.0, 37.7.

Pro:−50.%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=8.8 Hz), 7.84, (d, 2,J=7.7 Hz), 7.67 (d, 2, J=7.7 Hz), 7.54 (d, 2, J=9.1 Hz), 7.31-7.40 (m,4), 4.37, 4.29, 4.04 (m, 6), 3.45 (m, 2), 2.05 (m, 1), 1.96 (m, 3).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 205.4, 155.9, 154.8, 152.5, 145.6,144.4, 141.4, 127.7, 127.2,125.2, 125.0, 122.4,120.0, 69.0, 66.8, 55.9,47.4, 27.4, 23.7, 20.0.

Ser(tBu):−53%

¹H NMR (270 MHz, d₆-acetone): δ, 8.31 (m, 1), 7.84 (d, 2, J=7.7 Hz),7.69 (d, 2, J=7.5 Hz), 7.53 (d, 1, J=9.4 Hz), 7.42-7.31 (m, 5), 4.35 (m,4), 3.65 (m, 4), 2.93 (d, 1, J=3.3 Hz), 1.17 (m, 11), 0.92 (d, 1, J=6.6Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.1, 156.9, 156.7, 153.2, 145.1,142.1, 128.5, 128.4, 127.9, 126.1, 126.0, 120.7, 116.5, 79.2, 73.7,73.4, 69.2, 67.0, 66.9, 62.4, 61.7, 54.2, 51.3, 48.1, 48.0, 47.9, 27.2,19.2.

Thr(tBu):−45%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=9.1 Hz), 7.84 (d, 2,J=7.7 Hz), 7.68 (d, 2, J=7.3 Hz), 7.51 (d, 2, J=9.1 Hz), 7.28-7.49 (dt,4), 6.4 (d, 1, J=9.2 Hz), 4.46, 4.39, 4.24 (dd, d, m, 5, J=4.8 Hz, 10.9Hz, 7.3 Hz), 3.96-4.03 (m, 2), 1.21 (m, 12).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 205.4, 156.6, 155.9, 152.5, 145.6,144.4, 144.2, 141.3, 127.7, 127.1, 125.2, 122.3, 120.0, 73.7, 68.3,66.3, 65.9, 60.6, 55.4, 54.9, 54.1, 47.3, 18.7.

Trp:−64%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=9.1 Hz), 7.85 (d, 2,J=7.7 Hz), 7.69 (m, 3), 7.52 (d, 2, J=8.8 Hz), 7.24-7.42 (m, 2), 4.359m, 6), 3.08 (d, 2, J=4.0 Hz).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.4, 156.9, 156.4, 153.0, 146.1,144.1, 144.8, 141.8, 137.4, 128.3, 127.7, 126.0, 125.7, 124.2, 123.1,121.7, 120.6, 119.2, 119.0, 112.2, 70.8, 66.5, 51.5, 47.8, 27.5.

Tyr(tBu):−50%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=9.1 Hz), 7.84 (d, 2,J=7.7 Hz), 7.64 (d, 2, J=7.3 Hz), 7.52 (d, 2, J=9.1 Hz), 7.29-7.38 9m,6), 6.90 (d, 2, J=8.42 Hz), 4.14-4.45 (m, 7), 2.89 (m, 4), 1.24 (s, 10).

¹³C NMR (67.8 MHz, d₆-acetone): δ, 206.2, 156.9, 156.7, 155.1, 153.2,146.3, 145.0, 142.0, 133.3, 130.8, 130.5, 128.4, 127.9, 126.0, 124.7,123.1, 120.7, 78.4, 70.9, 66.9, 52.5, 52.4, 48.0, 37.0.

Val:−62%

¹H NMR (270 MHz, d₆-acetone): δ, 8.30 (d, 2, J=9.1 Hz), 7.83 (d, 2,J=7.3 Hz), 7.70 (d, 2, J=7.1 Hz), 7.54 (d, 2, J=9.2 Hz), 7.30-7.44 (m,4), 4.10-4.44 (m, 6), 3.89 (d, 1, J=6.2 Hz), 0.86 (m, 6).

¹³C NMR (67.8 MHz, d₆-acetone) δ NMR (67.8 MHz, d₆-acetone): d, 206.2,156.4, 155.8, 152.3, 146.1, 145.3, 141.9, 128.5, 127.9, 127.1, 125.9,123.1, 119.9, 71.2, 66.2, 48.1, 47.5, 24.3, 22.7.

What is claimed is:
 1. A compound of formula (I):

wherein: X is selected from COOH, NHZ, SH, or OH, where Z is either H oran amine protecting group; A is O, S, NH, or a single bond; R₂ and R₃are independently selected from: H, R, OH, OR, ═O, ═CH—R, ═CH₂,CH₂—CO₂R, CH₂—CO₂H, CH₂—SO₂R, O—SO₂R, CO₂R, COR, CN and there isoptionally a double bond between C1 and C2 or C2 and C3; R₆, R₇, and R₉are independently selected from H, R, OH, OR, halo, nitro, amino, andMe₃Sn; R₁₁ is either H or R; Q is S, O or NH; R₁₀ is a nitrogenprotecting group; where R is selected from the group consisting of alower alkyl group having 1 to 10 carbon atoms and optionally containsone or more carbon-carbon double or triple bonds, which may form part ofa conjugated system; an alkyl-C(O)-alkyl group having 3 to 10 carbonatoms; an alkyl-O-alkyl group having 2 to 10 carbon atoms; analkyl-S-alkyl group having 2 to 10 carbon atoms; an aralkyl group of upto 12 carbon atoms, wherein an alkyl group of the aralkyl groupoptionally contains one or more carbon-carbon double or triple bonds,which may form part of a conjugated system; and an aryl group of up to12 carbon atoms; and where R is optionally substituted by one or morehalo, hydroxy, amino, or nitro groups; and Y is a divalent groupselected from the group consisting of a lower alkylene group having 1 to10 carbon atoms, which optionally contains one or more carbon-carbondouble or triple bonds, which may form part of a conjugated system; analkylene-C(O)- alkylene group having 3 to 10 carbon atoms; analkylene-O-alkylene group having 2 to 10 carbon atoms; analkylene-S-alkylene group having 2 to 10 carbon atoms; an aralkylenegroup of up to 12 carbon atoms wherein an alkylene portion of thearalkylene group optionally contains one or more carbon-carbon double ortriple bonds which may form part of a conjugated system; or an arylenegroup of up to 12 carbon atoms; and wherein Y is optionally substitutedby one or more halo, hydroxy, amino or nitro groups.
 2. A compoundaccording to claim 1, wherein R and HY are independently selected fromlower alkyl group having 1 to 10 carbon atoms, or an aralkyl group, ofup to 12 carbon atoms, or an aryl group, of up to 12 carbon atoms,optionally substituted by one or more halo, hydroxy, amino, or nitrogroups.
 3. A compound according to claim 2, wherein R and HY areindependently selected from lower alkyl groups having 1 to 10 carbonatoms optionally substituted by one or more halo, hydroxy, amino, ornitro groups.
 4. A compound according to claim 3, wherein R or HY areindependently selected from unsubstituted straight or branched chainalkyl groups, having 1 to 10 carbon atoms.
 5. A compound according toclaim 1, wherein R₁₀ is selected from the group consisting of Fmoc,Nvoc, Teoc, Troc, Boc, CBZ, Alloc, and Psec.
 6. A compound according toclaim 1, wherein R₇ is selected from the group consisting of R, OH, ORand amino.
 7. A compound according to claim 1, wherein Q is 0, and/orR₁₁ is H.
 8. A compound according to claim 1, wherein R₆ and R₉ are H.9. A compound according to claim 8, wherein R₇ is an alkoxy group.
 10. Acompound according to claim 1, wherein R₂ and R₃ are H.
 11. A compoundaccording to claim 1, wherein there is no double bond between C2 and C3.12. A compound according to claim 1, wherein —Y—A— is an alkoxy chain.